Methods and compositions for generating complex trait loci

ABSTRACT

Compositions and methods are provided for stacking multiple independent transgenic loci into the genome of a plant. Compositions include plants, seeds or plant cells comprising at least one transgenic target site and at least one genomic locus of interest integrated at different genomic sites within a genomic window. Plant breeding techniques can be employed such that the transgenic target site and the genomic locus of interest can be bred together. In this way, multiple independent transgene integrations can be generated within a genomic window to create a complex trait locus. The complex trait locus is designed such that the transgenic target sites and/or genomic loci of interest can segregate independently of each other, thus providing the benefit of altering a complex trait locus by breeding-in and breeding-away specific elements. Various methods can also be employed to modify the target sites such that they contain a variety of polynucleotides of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.13/748,704 filed 24 Jan. 2013, now Allowed, which claims the benefit ofU.S. Provisional Application No. 61/591,329, filed Jan. 27, 2012, eachof which is hereby incorporated herein in its entirety by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file namedBB1920USCNT_SequenceListing.TXT, created on 22 Aug. 2019, and having asize of 11,935 bytes and is filed concurrently with the specification.The sequence listing contained in this ASCII formatted document is partof the specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The invention relates to the field of plant molecular biology. Inparticular, methods and compositions are provided for altering thegenome of a plant.

BACKGROUND OF THE INVENTION

Recombinant DNA technology has made it possible to insert foreign DNAsequences into the genome of an organism, thus, altering the organism'sphenotype. The most commonly used plant transformation methods areAgrobacterium infection and biolistic particle bombardment in whichtransgenes integrate into a plant genome in a random fashion and in anunpredictable copy number.

Unfortunately, the problems associated with these methods can result inreduced agronomics, additional costs for further research, creation ofadditional transgenic events, and slower time to product. Thus, moreefficient methods are needed for targeting the insertion of a sequenceof interest into a desirable genomic position, for readily modifying thetargeted polynucleotide and/or for stacking additional polynucleotidesof interest near the desired integration site.

BRIEF SUMMARY OF THE INVENTION

Methods and compositions for producing in a plant a complex trait locusin a genomic window comprising at least one transgenic target site andat least one genomic locus of interest are provided. The compositionsprovide a plant or seed having in its genome a genomic window of about10 cM in length wherein the genomic locus (loci) of interest, and thetransgenic target site(s) have different genomic insertion sites andsegregate independently from each other at a rate of about 10% to about0.1%. The transgenic target sites can comprise at least a firstrecombination site and a second recombination site and the first andsecond recombination sites are dissimilar with respect to one another.The transgenic target sites can further comprise a polynucleotide ofinterest and can be altered by site-specific integration methods.

Further provided is a method of producing a complex trait locus in thegenome of a plant comprising applying plant breeding techniques to afirst plant having in its genome a genomic window of about 10 cM with atleast a first transgenic target site. The method comprises breeding tosaid first plant a second plant comprising a first genomic locus ofinterest in the genomic window and selecting a progeny comprising saidfirst transgenic target site and said first genomic locus of interest,wherein said first transgenic target site and said first genomic locushave different genomic insertion sites in said progeny plant. Using suchmethods, various transgenic target sites and/or polynucleotides ofinterest can be introduced into a genomic window. Also provided aremethods of altering the complex trait locus by utilizing variousbreeding techniques or by employing site-specific recombinationtechniques to add, remove, or replace transgenic target sites, genomicloci of interest or polynucleotides of interest.

Additionally provided is a library of plants, seeds or plant cellscomprising a transgenic target site and methods of making the library.The library comprises a population of plants, seeds or plant cells eachcomprising a transgenic target site having a different genomic insertionsite and the transgenic target sites segregate independently from oneanother when combined into a single plant genome. Further provided aresubpopulations of the library wherein each member comprises a transgenictarget site with a different genomic insertion site within a givengenomic window and said transgenic target sites segregate independentlyat a rate of about 10% to about 0.1% when present in the same genome.The transgenic target sites of the library can be located at definedintervals throughout a given genomic window such that all possiblepositions for transgenic target site insertion within the given genomicwindow are represented by the members of a population of the library.Thus, breeding techniques can be applied to given subpopulations of thelibraries to produce a complex trait locus in a plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A and FIG. 1B depict a non-limiting example design of a complextrait locus generated by crossing and/or retransformation is shown. GIindicates gene of interest and TG denotes a transgene. A to D is a 10 cMgenomic region.

FIG. 2A depicts the T-DNA region of SSI platform II variants forpPHP35557 and FIG. 2B depicts the same for pPHP44290. These plasmids area derivative of an Agrobacterium binary vector intermediate related topSB11 used for transformation of corn published in Komari, et al., 1996.A selectable marker gene(s) is located near the T-DNA left bordersequence (LB). A multiple cloning site (not shown) (PHP35557) or aGateway destination site (PHP44290) for addition of trait genes is addednear the right T-DNA border (RB). The trait addition region is locatedbetween a pair of loxP sites for the capability to do CRE/lox excision.Site specific integration (SSI) capability is enabled by placing FRTsites, FRT1 and FRT87, around selectable marker gene(s).

FIG. 3 is a Schematic overview of plasmid PHP44556.

FIG. 4 is a Schematic overview of ligation-mediated nested PCR (LMnPCR).Genomic DNA was extracted from leaf tissue of T0 transgenic plantsderived from the transformation process and then randomly sheared usingmechanical force. DNA shearing generated large numbers of randomfragments, a small subset of which contained a portion of the newlyinserted transgene where it was contiguous with genomic DNA. The genomicDNA fragments with transgene sequence represent the genomic insertionsite and the insertion site flanking sequence (FS) is defined by theregion where genomic DNA is contiguous with transgene sequence. Adefined DNA linker was ligated to the ends of random genomic fragmentsto facilitate the use of PCR methods to amplify the relatively raretransgene insertion fragments. PCR was designed using primers thathybridized to the ends of the transgene and primers that hybridized tothe DNA linker. PCR bands that were amplified should contain FS and weresubmitted for sequence analysis and then confirmed. Confirmation ofsequence results involved BLAST analysis of the amplified PCR sequenceto the maize genome sequence database.

FIG. 5A depicts Transgenic target sites (TTS) and insertion site (IS)candidates for the complex trait locus CTL3A on maize chromosome 1. FIG.5B depicts Transgenic target sites (TTS) candidates for the complextrait locus CTL6A on maize chromosome 6.

FIG. 6 is a Schematic of the transgenic target sites (TTS) and insertionsites (IS) of the CTL3A complex trait locus in relation to the genomicwindow of interest (TRAIT3A) and public BACS on the maize physical map.

FIG. 7 depicts Site specific integration in transgenic maize eventcomprising a transgenic target site for SSI.

FIG. 8 depicts Recombinase mediated cassette exchange (RMCE) at TTS-3A2in complex trait locus CTL3A.

FIG. 9 is a Schematic of the transgenic target sites (TTS) of the CTL6Acomplex trait locus in relation to the genomic window of interest(TRAIT6A) and public BACS on the maize physical map.

FIG. 10 shows Expression analysis of several insertions at one genomicposition.

FIG. 11 is a Schematic map of the QC599A DNA fragment used for biolisticsoybean transformation to create transgenic SSI target events. FRT1 andFRT87 sites used for qPCR assays and three unique restriction sitesAflII, NsiI, PciI used for inverse PCR are marked. The GM-SAMS PRO hasan intron indicated by the solid line.

FIG. 12 is a Schematic description of FLP recombinase mediated cassetteexchange in soybean. Target DNA previously integrated in soybean genomerecombines with the donor DNA at both the FRT1 and FRT87 sites with thehelp of transiently expressed FLP recombinase. The target DNA cassetteflanked by the FRT1 and FRT87 sites is replaced by the donor DNAcassette flanked by the FRT1 and FRT87 sites resulting site-specificintegration of the donor cassette to the exact same genomic site of thetarget.

FIG. 13 depicts Identification of SSI target lines containing TransgenicTarget Sites. The genomic DNA of single copy target events wasseparately digested with three restriction enzymes AflII, NsiI, and PciIthat all cut the QC599A transgene only once and the nearby flankinggenomic border DNA, for example the PciI digestion. The resulting mixedgenomic border and QC599A transgene DNA fragment was circularized byself-ligation, PCR amplified, and sequenced. Two rounds of PCRamplifications using two sets of primers for each of the 5′ border and3′ border were used to specifically amplify the border-QC599A DNAfragments.

FIG. 14 depicts the genetic location of transgenic SSI target sitesTTS-LA1 and TTS-LA2 and one gene of interest (TRAITLA) in the genomicwindow comprising the complex trait locus CTL-LA on chromosome 19 insoybean. SSI target sites TTS-LA1 and TTS-LA2 were created independentlyand brought together by crossing.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which thisinvention pertains having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the invention is not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

I. Overview

Compositions and methods are provided for stacking multiple independenttransgenic loci into the genome of a plant. Compositions include plants,seeds or plant cells comprising at least one transgenic target site andat least one genomic locus of interest integrated at different genomicsites within a genomic window. Plant breeding techniques can be employedsuch that the transgenic target site and the at least one genomic locusof interest can be bred as a single complex trait locus. In this way,multiple independent transgene integrations can be generated within agenomic window to create a complex trait locus. As used herein, a“complex trait locus” (plural: “complex trait loci”) is a chromosomalsegment within a defined genomic window that comprises at least onetransgenic target site and at least one genomic locus of interest,wherein the target site and the genomic locus of interest have differentgenomic insertion sites within the defined genomic window. The complextrait locus is designed such that the transgenic target sites and/orgenomic loci of interest can segregate independently of each otherduring meiosis. This allows traits to be bred in and bred out of thecomplex trait locus. Thus, the methods described herein provide thebenefit of being able to alter a complex trait locus by breeding in andbreeding away specific elements of the complex trait locus. A variety ofmethods can also be employed to further modify the transgenic targetsites and/or genomic loci of interest such that they contain a varietyof polynucleotides of interest.

II. Compositions

A. Genomic Window

Provided herein is a plant or seed having in its genome a genomicwindow. As used herein, a “genomic window” is a segment of a chromosomein the genome of a plant that is desirable for producing a complex traitlocus or the segment of a chromosome comprising a complex trait locusthat was produced by the methods provided herein. The genomic window caninclude, for example, one or more traits prior to producing a complextransgenic trait locus therein. As used herein, a “trait” refers to thephenotype conferred from a particular gene or grouping of genes.

The genomic window can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15 or more centimorgans (cM) in length. Alternatively, thegenomic window can be about 1-10 cM, about 2-8 cM, about 2-5 cM, about3-10 cM, about 3-6 cM, about 4-10 cM, about 4-7 cM, about 5-10 cM, about5-8 cM, about 6-10 cM, about 6-9 cM, about 7-10 cM, about 8-10 cM orabout 9-10 cM in length. In one embodiment, the genomic window is about10 centimorgans (cM) in length or about 5 cM in length. A “centimorgan”(cM) or “map unit” is the distance between two linked genes, markers,target sites, genomic loci of interest, loci, or any pair thereof,wherein 1% of the products of meiosis are recombinant. Thus, acentimorgan is equivalent to a distance equal to a 1% averagerecombination frequency between the two linked genes, markers, targetsites, loci, genomic loci of interest or any pair thereof.

The genomic window can comprise various components. Such components caninclude, for example, transgenic target sites, native genes, genomicloci of interest, recombination sites and polynucleotides of interest.The genomic window can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more transgenic target sites such that each transgenic target sitehas a different genomic insertion site within the genomic window. Inaddition, the genomic window can comprise at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more genomic loci of interest each having a differentgenomic insertion site. By a “different genomic insertion site” is meantthat each component of the genomic window (i.e. transgenic target sitesand genomic loci of interest) is inserted into the genome at a differentlocation and as such each component can segregate independently from oneanother. For example, the genomic window can comprise a combination oftransgenic target sites and/or genomic loci of interest such that eachtarget site or genomic loci of interest has a different genomicinsertion site within the genomic window.

The components of the genomic windows provided herein have differentgenomic insertion sites and as such can segregate independently from oneanother. As used herein, “segregate independently”, is used to refer tothe genetic separation of any two or more genes, transgenes, nativegenes, mutated genes, target sites, genomic loci of interest, markersand the like from one another during meiosis. Assays to measure whethertwo genetic elements segregate independently are known in the art. Assuch, any two or more genes, transgenes, native genes, mutated genes,target sites, genomic loci of interest, markers and the like within agenomic window provided herein, have genomic insertion sites located atan appropriate distance from one another so that they generallysegregate independently at a rate of about 10% or less. Thus, thecomponents of the genomic windows provided herein can segregateindependently from one another at a rate of about 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or0.1%. Alternatively, the components of the genomic windows providedherein can segregate independently from one another at a rate of about10-0.1%, about 10-0.5%, about 10-1%, about 10-5%, about 9-0.1%, about9-0.5%, about 9-1%, about 9-5%, about 8-0.1%, about 8-0.5%, about 8-1%,about 8-4%, about 7-0.1%, about 7-0.5%, about 7-1%, about 7-4%, about6-0.1%, about 6-1%, about 6-0.5%, about 6-3%, about 5-0.1%, about 5-1%,about 5-0.5%, about 4-0.1%, about 4-1%, about 4-0.5%, about 3-0.1%,about 3-1%, about 3-0.5%, about 2-0.1%, about 2-0.5%, about 1-0.1% orabout 1-0.5%. For example, if the genomic window comprises a transgenictarget site and a genomic locus of interest that are about 5 cM fromeach other, the transgenic target site and the genomic locus of interestwould segregate independently at a rate of about 5%.

In one embodiment, the genomic window comprises at least a firsttransgenic target site, a second transgenic target site, and at leastone genomic locus of interest wherein each of the transgenic targetsites and genomic loci of interest have a different genomic insertionsite and segregate independently from one another at a rate of about 10%to about 0.1%.

Any given genomic window can also comprise at least one altered targetsequence that originated from a corresponding target sequence that wasrecognized and cleaved by a double-strand break-inducing agent. As usedherein, a “double-strand-break-inducing agent” refers to any nucleasewhich produces a double-strand break in the target sequence. A “targetsequence” refers to a polynucleotide sequence in the genome of a plantcell that comprises a recognition sequence for adouble-strand-break-inducing agent at which a double-strand-break isinduced. An “altered target sequence” refers to a target sequencecomprising at least one alteration when compared to a non-altered targetsequence. “Alterations” can include, for example: (i) replacement of atleast one nucleotide, (ii) a deletion of at least one nucleotide, (iii)an insertion of at least one nucleotide, or (iv) any combination of(i)-(iii). As such, insertion sites for the various components of thegenomic window (i.e. transgenic target sites or genomic loci ofinterest) can be created in any given genomic window. Methods ofcreating altered target sequences are known and are disclosed in U.S.Provisional Application No. 61/466,602, filed on Mar. 23, 2011, which isherein incorporated by reference in its entirety.

In specific embodiments, the genomic window is flanked by at least afirst marker and a second marker. Non-limiting examples of such markerson chromosome 1 of corn include, for example, UMC1160, UMC2224, NP1579B,PMCB1, IDP3917, GPM199C, IDP1425, MMP68, UMC2225, STD2C(DBA), TIDP3300,CSU1171, SUT1, UMC1166, AY107207, UMC1568, IDP3783, BNLG1429, IDP209,LTK1 and IDP7169. Table 2 depicts the public IBM2 genetic map positionsfor markers on chromosome 1 of corn. Non-limiting examples of markers onchromosome 6 of corn include, for example, UMC1625, UMC2196, UMC2312,BNLG1867, PZA03047, UMC1229, UCK1, RZ390D(CYB5), MMP20, MMP10, MMP160,PHP20528, UMC2314, UAZ232B(SCI), UMC2313, CD0545, PHP20854, UMC1133,UFG69, MMP76, Y1, BNLG1422, MMP108B, MMP4, UMC1006 and RZ444E. Table 6 Adepicts the public IBM2 genetic map positions for markers on chromosome6 of corn. Non-limiting examples of such markers on chromosome 19 ofsoybean include, for example, SATT613, SATT284, S60414-TB, SATT462,SATT481, SATT156 and SCT_010. Table 11 depicts the public genetic mappositions for markers on chromosome 19 of soybean.

B. Components of the Genomic Window

i. Trans genic Target Sites and Methods of Altering

A transgenic target site can comprise various components. As usedherein, by “target site” is intended a polynucleotide comprising anucleotide sequence comprising at least one recombination site. By“transgenic target site” is meant a target site that is non-nativeeither in sequence and/or in genomic location to the plant genome. Insome embodiments, the transgenic target site can comprise at least 1, 2,3, 4, 5, 6 or more recombination sites for site-specific recombination.In one embodiment, the transgenic target site comprises a firstrecombination site and a second recombination site. In such embodiments,the first and second recombination sites may be dissimilar with respectto one another or may be dissimilar and have a decreased compatibilitywith respect to one another. Such first and second recombination sitesare able to recombine with their corresponding or identicalrecombination site when provided with the appropriate recombinase.

One or more intervening sequences may be present between therecombination sites of the target site. Intervening sequences ofparticular interest would include linkers, adapters, selectable markers,polynucleotides of interest, other recombination sites, promoters and/orother sites that aid in vector construction or analysis. Variouspolynucleotides of interest then could be employed between therecombination sites. Methods of altering the target sites are discussedin greater detail elsewhere herein. In addition, the recombination sitesof the target site can be located in various positions, including, forexample, within intronic sequences, coding sequences, or untranslatedregions.

The recombination sites employed in the methods and compositionsprovided herein can be “corresponding” sites or “dissimilar” sites. By“corresponding recombination sites” or a “set of correspondingrecombination sites” is intended that the recombination sites have thesame or corresponding nucleotide sequence. A set of correspondingrecombination sites, in the presence of the appropriate recombinase,will efficiently recombine with one another (i.e., the correspondingrecombination sites are recombinogenic).

In other embodiments, the recombination sites are dissimilar. By“dissimilar recombination sites” or a “set of dissimilar recombinationsites” is intended that the recombination sites are distinct (i.e., haveat least one nucleotide difference).

The recombination sites within “a set of dissimilar recombination sites”can be either recombinogenic with respect to one another or have adecreased compatibility with respect to one other. By “recombinogenic”is intended that the set of recombination sites are capable ofrecombining with one another.

In other embodiments, a set of dissimilar recombination sites cancomprise sets of recombination sites having a decreased compatibilitywith respect to one another. By “decreased compatibility” is intendedthe set of recombination sites, in the presence of the appropriaterecombinase, will have a decreased efficiency of recombination comparedto that seen with their cognate site. In some embodiments, having adecrease in compatibility will result in no recombination between thesites. In other embodiments, having a decrease in compatibility willresult in a minimal level of recombination between the sites. Thus,suitable recombination sites having a decrease in compatibility with oneanother for use in the methods and compositions provided herein includethose sites that recombine (or excise) with one another at a frequencylower than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 4%, or3%. In some embodiments the recombination sites having a decrease incompatibility with one another recombine (or excise) with one another ata frequency lower than the detectable limit under standard conditions inan excision assay, lower than 2%, 1.5%, 1%, 0.75%, 0.5%, 0.25%, 0.1%,0.075, 0.005%, 0.001%. Each recombination site within the “set ofdissimilar-recombination sites” is biologically active and therefore canrecombine with an identical site.

In some embodiments, the genomic window comprises a first transgenictarget site comprising a first and a second recombination site that aredissimilar with respect to one another and a second transgenic targetsite comprising a third and a fourth recombination site that aredissimilar with respect to one another. In other embodiments, thegenomic window comprises a first transgenic target site comprising afirst and a second recombination site that are dissimilar and have adecreased compatibility with respect to one another and a secondtransgenic target site comprising a third and a fourth recombinationsite that are dissimilar and have a decreased compatibility with respectto one another. In some cases, the first transgenic target site and thesecond transgenic target site segregate independently from one anotherat a rate of about 5% to about 0.1%. Thus, any of the various targetsites provided herein can segregate independently from one another at arate of about 10-0.1%, about 10-0.5%, about 10-1%, about 10-5%, about9-0.1%, about 9-0.5%, about 9-1%, about 9-5%, about 8-0.1%, about8-0.5%, about 8-1%, about 8-4%, about 7-0.1%, about 7-0.5%, about 7-1%,about 7-4%, about 6-0.1%, about 6-0.5%, about 6-1%, about 6-3%, about5-0.1%, about 5-0.5%, about 5-1%, about 4-0.1%, about 4-0.05%, about4-1%, about 3-0.1%, about 3-0.5%, about 3-1%, about 2-0.1%, about2-0.5%, about 1-0.1% or about 1-0.5%. The various target sites providedherein can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2,3, 4, 5, 6, 7, 8, 9, 10 cM or more from one another in the genomicwindow. Alternatively, the various target sites can be about 0.5-10 cM,about 1-10 cM, about 2-10 cM, about 2-5 cM, about 3-10 cM, about 3-6 cM,about 4-10 cM, about 4-7 cM, about 5-10 cM, about 5-8 cM, about 6-10 cM,about 6-9 cM, about 7-10 cM, about 8-10 cM, about 9-10 cM, about 0.1-0.5cM, about 0.1-1 cM, about 0.1-2 cM, about 0.1-3 cM, about 0.1-4 cM,about 0.1-5 cM, about 0.1-6 cM, about 0.1-7 cM about 0.1-8 cM, about0.1-9 cM or about 0.1-10 cM from one another in the genomic window.

In a specific embodiment, the recombination sites of the secondtransgenic target site are different from the dissimilar sites of thefirst transgenic target site. Alternatively, the second transgenictarget site can comprise the same dissimilar sites as the firsttransgenic target site. In a further embodiment, the genomic windowcomprises a third transgenic target site comprising a fifth and a sixthrecombination site that are dissimilar with respect to one another. Inyet a further embodiment, the genomic window comprises a thirdtransgenic target site comprising a fifth and sixth recombination sitesthat are dissimilar and have a decreased compatibility with respect toone another. In all such cases, the first, second and third transgenictarget sites have different genomic insertion sites.

Various recombination sites can be employed in the methods andcompositions provided herein (i.e. in the various transgenic targetsites or genomic loci of interest disclosed herein). By “recombinationsite” is intended a recombination site and active variants thereof. Manyrecombination systems are known in the art and one of skill willrecognize the appropriate recombination site to be used with therecombination system of interest. As discussed in greater detailelsewhere herein, various combinations of recombination sites can beemployed including, sets of dissimilar sites and correspondingrecombination sites and/or dissimilar recombination sites and/or sitesthat are dissimilar and have a decreased compatibility with respect toone another can be used in the various methods and compositions providedherein. Accordingly, any suitable recombination site or set ofrecombination sites may be utilized herein, including a FRT site, abiologically active variant of a FRT site (i.e. a mutant FRT site), aLOX site, a biologically active variant of a LOX site (i.e. a mutant LOXsite), any combination thereof, or any other combination ofrecombination sites known in the art. Examples of FRT sites include, forexample, the wild type FRT site (FRT1) (SEQ ID NO: 1), and variousmutant FRT sites, including but not limited to, FRT5 (SEQ ID NO: 2),FRT6 (SEQ ID NO: 3), FRT7 (SEQ ID NO: 4), FRT12 (SEQ ID NO: 5) and FRT87(SEQ ID NO: 6). See, for example, U.S. Pat. No. 6,187,994. See also, USPublication No. 2011-0047655, herein incorporated by reference.

Recombination sites from the Cre/Lox site-specific recombination systemcan also be used. Such recombination sites include, for example, wildtype LOX sites and mutant LOX sites. An analysis of the recombinationactivity of mutant LOX sites is presented in Lee et al. (1998) Gene216:55-65, herein incorporated by reference. Also, see for example,Schlake and Bode (1994) Biochemistry 33:12746-12751; Huang et al. (1991)Nucleic Acids Research 19:443-448; Sadowski (1995) In Progress inNucleic Acid Research and Molecular Biology Vol. 51, pp. 53-91; Cox(1989) In Mobile DNA, Berg and Howe (eds) American Society ofMicrobiology, Washington D.C., pp. 116-670; Dixon et al. (1995) Mol.Microbiol. 18:449-458; Umlauf and Cox (1988) EMBO 7:1845-1852; Buchholzet al. (1996) Nucleic Acids Research 24:3118-3119; Kilby et al. (1993)Trends Genet. 9:413-421; Rossant and Geagy (1995) Nat. Med. 1: 592-594;Albert et al. (1995) The Plant J. 7:649-659; Bayley et al. (1992) PlantMol. Biol. 18:353-361; Odell et al. (1990) Mol. Gen. Genet. 223:369-378;Dale and Ow (1991) Proc. Natl. Acad. Sci. USA 88:10558-10562; Qui et al.(1994) Proc. Natl. Acad. Sci. USA 91:1706-1710; Stuurman et al. (1996)Plant Mol. Biol. 32:901-913; Dale et al. (1990) Gene 91:79-85; Albert etal. (1995) The Plant J. 7:649-659 and WO 01/00158; all of which areherein incorporated by reference.

In a specific embodiment, at least one of the first and the secondrecombination sites comprises FRT1 (SEQ ID NO: 1), FRT5 (SEQ ID NO: 2),FRT6 (SEQ ID NO: 3), FRT7 (SEQ ID NO: 4), FRT12 (SEQ ID NO: 5) or FRT87(SEQ ID NO: 6). In a specific embodiment, the first and the secondrecombination sites of the target site comprise a FRT1 site and a FRT87site.

Active variants and fragments of recombination sites (i.e. SEQ ID NOS:1-6) are also encompassed by the compositions and methods providedherein. Fragments of a recombination site retain the biological activityof the recombination site and hence facilitate a recombination event inthe presence of the appropriate recombinase. Thus, fragments of arecombination site may range from at least about 5, 10, 15, 20, 25, 30,35, 40 nucleotides, and up to the full-length of a recombination site.Active variants can comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thenative recombination site, wherein the active variants retain biologicalactivity and hence facilitate a recombination event in the presence ofthe appropriate recombinase. Assays to measure the biological activityof recombination sites are known in the art. See, for example, Senecollet al. (1988) J. Mol. Biol. 201:406-421; Voziyanov et al. (2002) NucleicAcid Research 30:7, U.S. Pat. No. 6,187,994, WO/01/00158, and Albert etal. (1995) The Plant Journal 7:649-659.

Recombinases are also employed in the methods and compositions providedherein. By “recombinase” is intended a native polypeptide that catalyzessite-specific recombination between compatible recombination sites. Forreviews of site-specific recombinases, see Sauer (1994) Current Opinionin Biotechnology 5:521-527; and Sadowski (1993) FASEB 7:760-767; thecontents of which are incorporated herein by reference. The recombinaseused in the methods can be a naturally occurring recombinase or abiologically active fragment or variant of the recombinase. Recombinasesuseful in the methods and compositions include recombinases from theIntegrase and Resolvase families, biologically active variants andfragments thereof, and any other naturally occurring or recombinantlyproduced enzyme or variant thereof that catalyzes conservativesite-specific recombination between specified DNA recombination sites.

The Integrase family of recombinases has over one hundred members andincludes, for example, FLP, Cre, Int, and R. For other members of theIntegrase family, see for example, Esposito et al. (1997) Nucleic AcidResearch 25:3605-3614 and Abremski et al. (1992) Protein Engineering5:87-91, both of which are herein incorporated by reference. Otherrecombination systems include, for example, the streptomycetebacteriophage phi C31 (Kuhstoss et al. (1991) J. Mol. Biol. 20:897-908);the SSV1 site-specific recombination system from Sulfolobus shibatae(Maskhelishvili et al. (1993) Mol. Gen. Genet. 237:334-342); and aretroviral integrase-based integration system (Tanaka et al. (1998) Gene17:67-76). In other embodiments, the recombinase is one that does notrequire cofactors or a supercoiled substrate. Such recombinases includeCre (SEQ ID NO: 7), FLP (SEQ ID NO: 8), or active variants or fragmentsthereof (SEQ ID NOS: 9 and 10).

The FLP recombinase is a protein that catalyzes a site-specific reactionthat is involved in amplifying the copy number of the two-micron plasmidof S. cerevisiae during DNA replication. As used herein, FLP recombinaserefers to a recombinase that catalyzes site-specific recombinationbetween two FRT sites. The FLP protein has been cloned and expressed.See, for example, Cox (1993) Proc. Natl. Acad. Sci. U.S.A. 80:4223-4227.The FLP recombinase for use in the methods and with the compositions maybe derived from the genus Saccharomyces. One can also synthesize apolynucleotide comprising the recombinase using plant-preferred codonsfor optimal expression in a plant of interest. A recombinant FLP enzymeencoded by a nucleotide sequence comprising maize preferred codons(FLPm) (SEQ ID NO: 10) that catalyzes site-specific recombination eventsis known. See, for example, U.S. Pat. No. 5,929,301, herein incorporatedby reference. Additional functional variants and fragments of FLP areknown. See, for example, Buchholz et al. (1998) Nat. Biotechnol.16:617-618, Hartung et al. (1998) J. Biol. Chem. 273:22884-22891, Saxenaet al. (1997) Biochim Biophys Acta 1340(2):187-204, and Hartley et al.(1980) Nature 286:860-864, all of which are herein incorporated byreference.

The bacteriophage recombinase Cre catalyzes site-specific recombinationbetween two lox sites. The Cre recombinase is known in the art. See, forexample, Guo et al. (1997) Nature 389:40-46; Abremski et al. (1984) J.Biol. Chem. 259:1509-1514; Chen et al. (1996) Somat. Cell Mol. Genet.22:477-488; Shaikh et al. (1977) J. Biol. Chem. 272:5695-5702; and,Buchholz et al. (1998) Nat. Biotechnol. 16:617-618, all of which areherein incorporated by reference. The Cre polynucleotide sequences mayalso be synthesized using plant-preferred codons. Such sequences (moCre)are described in WO 99/25840, herein incorporated by reference and setforth in SEQ ID NO: 9.

It is further recognized that a chimeric recombinase can be used in themethods. By “chimeric recombinase” is intended a recombinant fusionprotein which is capable of catalyzing site-specific recombinationbetween recombination sites that originate from different recombinationsystems. That is, if a set of functional recombination sites,characterized as being dissimilar with respect to one another, isutilized in the methods and compositions and comprises a FRT site and aLoxP site, a chimeric FLP/Cre recombinase or active variant or fragmentthereof will be needed or, alternatively, both recombinases may beseparately provided. Methods for the production and use of such chimericrecombinases or active variants or fragments thereof are described in WO99/25840, herein incorporated by reference.

By utilizing various combinations of recombination sites in thetransgenic target sites provided herein, the methods provide a mechanismfor the site-specific integration of polynucleotides of interest into aspecific site in the plant genome. The methods also allow for thesubsequent insertion of additional polynucleotides of interest into thespecific genomic site.

As used herein, by “providing” is intended any method that allows for anamino acid sequence and/or a polynucleotide to be brought together withthe recited components. A variety of methods are known in the art forthe introduction of nucleotide sequence into a plant. Any means can beused to bring together the various components of the recombinationsystem (i.e., the transgenic target site and the appropriaterecombinase), including, for example, transformation and sexualcrossing. See, also, WO99/25884 herein incorporated by reference. Inaddition, the recombinase may be provided by the introduction of thepolypeptide or mRNA into the cell.

Active variants and fragments of recombinases (i.e. FLP or Cre) are alsoencompassed by the compositions and methods provided herein. Such activevariants can comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thenative recombinase, wherein the active variants retain biologicalactivity and hence implement a recombination event. Assays forrecombinase activity are known and generally measure the overallactivity of the enzyme on DNA substrates containing recombination sites.For example, to assay for FLP activity, inversion of a DNA sequence in acircular plasmid containing two inverted FRT sites can be detected as achange in position of restriction enzyme sites. This assay is describedin Vetter et al. (1983) PNAS 80:7284. Alternatively, excision of DNAfrom a linear molecule or intermolecular recombination frequency inducedby the enzyme may be assayed, as described, for example, in Babineau etal. (1985) Journal of Biological Chemistry 260:12313; Meyer-Leon et al.(1987) Nucleic Acid Res 15:6469; and Gronostajski et al. (1985) Journalof Biological Chemistry 260:12328. Alternatively, recombinase activitymay also be assayed by excision of a sequence flanked by recombinogenicFRT sites that upon removal will activate an assayable marker gene.

ii. Genomic Locus of Interest

As used herein, a “genomic locus of interest” comprises a collection ofspecific polymorphisms that are inherited together. A given genomiclocus can comprise, but is not limited to, a transgene, a native gene oran additional transgenic target site that can comprise dissimilar pairsof recombination sites or pairs of recombination sites that aredissimilar and have a decreased compatibility with respect to oneanother.

The genomic locus of interest can be, for example, any modification thatconfers a trait, such as a transgene or a native trait. In oneembodiment, the genomic locus of interest comprises a native trait. Asused herein, a “native trait” refers to a trait found in nature. Inanother embodiment, the genomic locus of interest comprises a transgene.

The number of genomic loci of interest that could be crossed into agenomic window of a plant is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore. Any desired trait can be introduced into the genome at a givengenomic locus of interest. Such traits include, but are not limited to,traits conferring insect resistance, disease resistance, herbicidetolerance, male sterility, abiotic stress tolerance, altered phosphorus,altered antioxidants, altered fatty acids, altered essential aminoacids, altered carbohydrates, or sequences involved in site-specificrecombination.

In specific embodiments, a given genomic locus of interest is associatedwith a desirable and/or favorable phenotype in a plant. For example,traits that confer insect resistance, disease resistance or herbicidetolerance would be desirable in a plant. In other embodiments, thegenomic locus is not associated with traits that affect the agronomiccharacteristics of the plant.

A given genomic locus of interest has its own genomic insertion sitewithin the genomic window. For example, a genomic locus of interest anda transgenic target site within the genomic window will have differentgenomic insertion sites within the genome. A given transgenic targetsite can be found within about 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4cM, 3 cM, 2 cM, 1 cM, 0.9 cM, 0.8 cM, 0.7 cM, 0.6 cM, 0.5 cM, 0.4 cM,0.3 cM, 0.2 cM, or 0.1 cM from the genomic locus of interest such thatthe target site and genomic locus of interest have different genomicinsertion sites. Alternatively, a given transgenic target site can befound within about 0.5-10 cM, about 1-10 cM, about 2-10 cM, about 2-5cM, about 3-10 cM, about 3-6 cM, about 4-10 cM, about 4-7 cM, about 5-10cM, about 5-8 cM, about 6-10 cM, about 6-9 cM, about 7-10 cM, about 8-10cM, about 9-10 cM, about 0.1-0.5 cM, about 0.1-1 cM, about 0.1-2 cM,about 0.1-3 cM, about 0.1-4 cM, about 0.1-5 cM, about 0.1-6 cM, about0.1-7 cM about 0.1-8 cM, about 0.1-9 cM or about 0.1-10 cM from thegenomic locus of interest such that the target site and genomic locus ofinterest have different genomic insertion sites. In a specificembodiment, a first transgenic target site or a second transgenic targetsite is found within about 5 cM from the genomic locus of interest. Inyet another embodiment, the first or second transgenic target site isfound within 2 cM or 1 cM from the genomic locus of interest. In suchcases where the genomic window comprises a third transgenic target site,the third transgenic target site can be found within about 5 cM from thegenomic locus of interest.

In some embodiments, the first transgenic target site and the secondtransgenic target site segregate independently from the genomic locus ofinterest at a rate of about 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%,0.5%, 0.4%, 0.3%, 0.2% or 0.1%. Alternatively, the first transgenictarget site and the second transgenic target site segregateindependently from the genomic locus of interest at a rate of about5-0.1%, about 5-1%, about 5-0.5%, about 4-0.1%, about 4-0.5%, about4-1%, about 3-0.1%, about 3-0.05%, about 3-1%, about 2-0.1%, about2-0.5%, about 1-0.1% or about 1-0.5%.

C. Polynucleotides of Interest

Any polynucleotide of interest (i.e., the “polypeptide of interest” or“gene of interest”) may be provided to the plant cells in the transgenictarget sites or genomic loci of interest of the methods and compositionsdisclosed herein either by transformation methods or breeding methodsdiscussed elsewhere herein. It is recognized that any polynucleotide ofinterest can be provided, integrated into the plant genome within agenomic window and expressed in a plant. The polynucleotide of interestor gene of interest can comprise, for example, a transgene or a nativegene. The methods disclosed herein, provide for at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more polynucleotides of interest to be integrated intoa genomic window.

Various changes in phenotype are of interest, including modifying thefatty acid composition in a plant, altering the amino acid content of aplant, altering a plant's pathogen defense mechanism, and the like.These results can be achieved by providing expression of heterologousproducts (i.e. polynucleotides of interest) or increased expression ofendogenous products in plants. Alternatively, the results can beachieved by providing for a reduction of expression of one or moreendogenous products, particularly enzymes or cofactors in the plant.These changes result in a change in phenotype of the transformed plant.

Polynucleotides of interest are reflective of the commercial markets andinterests of those involved in the development of the crop. Crops andmarkets of interest change, and as developing nations open up worldmarkets, new crops and technologies will emerge also. In addition, asour understanding of agronomic traits and characteristics such as yieldand heterosis increase, the choice of genes for transformation willchange accordingly. General categories of genes of interest include, forexample, those genes involved in information, such as zinc fingers,those involved in communication, such as kinases, and those involved inhousekeeping, such as heat shock proteins. More specific categories oftransgenes, for example, include genes encoding important traits foragronomics, insect resistance, disease resistance, herbicide resistance,sterility, grain characteristics, and commercial products. Genes ofinterest include, generally, those involved in oil, starch,carbohydrate, or nutrient metabolism as well as those affecting kernelsize, sucrose loading, and the like.

Polynucleotides/polypeptides of interest include, but are not limitedto, herbicide-tolerance coding sequences, insecticidal coding sequences,nematicidal coding sequences, antimicrobial coding sequences, antifungalcoding sequences, antiviral coding sequences, abiotic and biotic stresstolerance coding sequences, or sequences modifying plant traits such asyield, grain quality, nutrient content, starch quality and quantity,nitrogen fixation and/or utilization, and oil content and/orcomposition. More specific polynucleotides of interest include, but arenot limited to, genes that improve crop yield, polypeptides that improvedesirability of crops, genes that alter phosphorus content, such as aphytase-encoding gene, genes that alter antioxidant content orcomposition, such as those that alter tocopherol and tocotrienolcontent, genes that alter carbohydrates, genes encoding proteinsconferring resistance to abiotic stress, such as drought, nitrogen,temperature, salinity, toxic metals or trace elements, or thoseconferring resistance to toxins such as pesticides and herbicides, or tobiotic stress, such as attacks by fungi, viruses, bacteria, insects, andnematodes, and development of diseases associated with these organisms.

Polynucleotides of interest can also be genes that create a site forsite-specific DNA integration. This includes the introduction of FRTsites that may be used in the FLP/FRT system and/or LOX sites that maybe used in the Cre/LoxP system. These systems and others are describedin detail elsewhere herein.

An “herbicide resistance protein” or a protein resulting from expressionof an “herbicide resistance-encoding nucleic acid molecule” includesproteins that confer upon a cell the ability to tolerate a higherconcentration of an herbicide than cells that do not express theprotein, or to tolerate a certain concentration of an herbicide for alonger period of time than cells that do not express the protein.Herbicide resistance traits may be introduced into plants by genescoding for resistance to herbicides that act to inhibit the action ofacetolactate synthase (ALS), in particular the sulfonylurea-typeherbicides, genes coding for resistance to herbicides that act toinhibit the action of glutamine synthase, such as phosphinothricin orbasta (e.g., the bar gene), glyphosate (e.g., the EPSP synthase gene andthe GAT gene), HPPD inhibitors (e.g., the HPPD gene) or other such genesknown in the art. See, for example, U.S. Pat. Nos. 7,626,077, 5,310,667,5,866,775, 6,225,114, 6,248,876, 7,169,970, 6,867,293, and U.S.Provisional Application No. 61/401,456, each of which is hereinincorporated by reference.

Agronomically important traits such as oil, starch, and protein contentcan be genetically altered in addition to using traditional breedingmethods. Modifications include increasing content of oleic acid,saturated and unsaturated oils, increasing levels of lysine and sulfur,providing essential amino acids, and also modification of starch.Hordothionin protein modifications are described in U.S. Pat. Nos.5,703,049, 5,885,801, 5,885,802, and 5,990,389, herein incorporated byreference. Another example is lysine and/or sulfur rich seed proteinencoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016,and the chymotrypsin inhibitor from barley, described in Williamson etal. (1987) Eur. J. Biochem. 165:99-106, the disclosures of which areherein incorporated by reference.

Commercial traits can also be encoded on a polynucleotide of interestthat could increase for example, starch for ethanol production, orprovide expression of proteins. Another important commercial use oftransformed plants is the production of polymers and bioplastics such asdescribed in U.S. Pat. No. 5,602,321. Genes such as β-Ketothiolase,PHBase (polyhydroxyburyrate synthase), and acetoacetyl-CoA reductase(see Schubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitateexpression of polyhyroxyalkanoates (PHAs).

Derivatives of the coding sequences can be made by site-directedmutagenesis to increase the level of preselected amino acids in theencoded polypeptide. For example, the gene encoding the barley highlysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor,U.S. application Ser. No. 08/740,682, filed Nov. 1, 1996, and WO98/20133, the disclosures of which are herein incorporated by reference.Other proteins include methionine-rich plant proteins such as fromsunflower seed (Lilley et al. (1989) Proceedings of the World Congresson Vegetable Protein Utilization in Human Foods and Animal Feedstuffs,ed. Applewhite (American Oil Chemists Society, Champaign, Ill.), pp.497-502; herein incorporated by reference); corn (Pedersen et al. (1986)J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; both ofwhich are herein incorporated by reference); and rice (Musumura et al.(1989) Plant Mol. Biol. 12:123, herein incorporated by reference). Otheragronomically important genes encode latex, Floury 2, growth factors,seed storage factors, and transcription factors.

Polynucleotides that improve crop yield include dwarfing genes, such asRht1 and Rht2 (Peng et al. (1999) Nature 400:256-261), and those thatincrease plant growth, such as ammonium-inducible glutamatedehydrogenase. Polynucleotides that improve desirability of cropsinclude, for example, those that allow plants to have reduced saturatedfat content, those that boost the nutritional value of plants, and thosethat increase grain protein. Polynucleotides that improve salt toleranceare those that increase or allow plant growth in an environment ofhigher salinity than the native environment of the plant into which thesalt-tolerant gene(s) has been introduced.

Sterility genes can also be encoded in an expression cassette andprovide an alternative to physical detasseling. Examples of genes usedin such ways include male tissue-preferred genes and genes with malesterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210.Other genes include kinases and those encoding compounds toxic to eithermale or female gametophytic development.

Polynucleotides/polypeptides that influence amino acid biosynthesisinclude, for example, anthranilate synthase (AS; EC 4.1.3.27) whichcatalyzes the first reaction branching from the aromatic amino acidpathway to the biosynthesis of tryptophan in plants, fungi, andbacteria. In plants, the chemical processes for the biosynthesis oftryptophan are compartmentalized in the chloroplast. See, for example,US Pub. 20080050506, herein incorporated by reference. Additionalsequences of interest include Chorismate Pyruvate Lyase (CPL) whichrefers to a gene encoding an enzyme which catalyzes the conversion ofchorismate to pyruvate and pHBA. The most well characterized CPL genehas been isolated from E. coli and bears the GenBank accession numberM96268. See, U.S. Pat. No. 7,361,811, herein incorporated by reference.

The polynucleotide sequences of interest may encode proteins involved inproviding disease or pest resistance. By “disease resistance” or “pestresistance” is intended that the plants avoid the harmful symptoms thatare the outcome of the plant-pathogen interactions. Pest resistancegenes may encode resistance to pests that have great yield drag such asrootworm, cutworm, European Corn Borer, and the like. Disease resistanceand insect resistance genes such as lysozymes or cecropins forantibacterial protection, or proteins such as defensins, glucanases orchitinases for antifungal protection, or Bacillus thuringiensisendotoxins, protease inhibitors, collagenases, lectins, or glycosidasesfor controlling nematodes or insects are all examples of useful geneproducts. Genes encoding disease resistance traits includedetoxification genes, such as against fumonosin (U.S. Pat. No.5,792,931); avirulence (avr) and disease resistance (R) genes (Jones etal. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; andMindrinos et al. (1994) Cell 78:1089); and the like.

Furthermore, it is recognized that the polynucleotide of interest mayalso comprise antisense sequences complementary to at least a portion ofthe messenger RNA (mRNA) for a targeted gene sequence of interest.Antisense nucleotides are constructed to hybridize with thecorresponding mRNA. Modifications of the antisense sequences may be madeas long as the sequences hybridize to and interfere with expression ofthe corresponding mRNA. In this manner, antisense constructions having70%, 80%, or 85% sequence identity to the corresponding antisensesequences may be used. Furthermore, portions of the antisensenucleotides may be used to disrupt the expression of the target gene.Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200nucleotides, or greater may be used.

In addition, the polynucleotide of interest may also be used in thesense orientation to suppress the expression of endogenous genes inplants. Methods for suppressing gene expression in plants usingpolynucleotides in the sense orientation are known in the art. Themethods generally involve transforming plants with a DNA constructcomprising a promoter that drives expression in a plant operably linkedto at least a portion of a nucleotide sequence that corresponds to thetranscript of the endogenous gene. Typically, such a nucleotide sequencehas substantial sequence identity to the sequence of the transcript ofthe endogenous gene, generally greater than about 65% sequence identity,about 85% sequence identity, or greater than about 95% sequenceidentity. See, U.S. Pat. Nos. 5,283,184 and 5,034,323; hereinincorporated by reference.

The polynucleotide of interest can also be a phenotypic marker. Aphenotypic marker is screenable or a selectable marker that includesvisual markers and selectable markers whether it is a positive ornegative selectable marker. Any phenotypic marker can be used.Specifically, a selectable or screenable marker comprises a DNA segmentthat allows one to identify, or select for or against a molecule or acell that contains it, often under particular conditions. These markerscan encode an activity, such as, but not limited to, production of RNA,peptide, or protein, or can provide a binding site for RNA, peptides,proteins, inorganic and organic compounds or compositions and the like.

Examples of selectable markers include, but are not limited to, DNAsegments that comprise restriction enzyme sites; DNA segments thatencode products which provide resistance against otherwise toxiccompounds including antibiotics, such as, spectinomycin, ampicillin,kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO) andhygromycin phosphotransferase (HPT)); DNA segments that encode productswhich are otherwise lacking in the recipient cell (e.g., tRNA genes,auxotrophic markers); DNA segments that encode products which can bereadily identified (e.g., phenotypic markers such as β-galactosidase,GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan(CFP), yellow (YFP), red (RFP), and cell surface proteins); thegeneration of new primer sites for PCR (e.g., the juxtaposition of twoDNA sequence not previously juxtaposed), the inclusion of DNA sequencesnot acted upon or acted upon by a restriction endonuclease or other DNAmodifying enzyme, chemical, etc.; and, the inclusion of a DNA sequencesrequired for a specific modification (e.g., methylation) that allows itsidentification.

Additional selectable markers include genes that confer resistance toherbicidal compounds, such as glufosinate ammonium, bromoxynil,imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See for example,Yarranton, (1992) Curr Opin Biotech 3:506-11; Christopherson et al.,(1992) Proc. Natl. Acad. Sci. USA 89:6314-8; Yao et al., (1992) Cell71:63-72; Reznikoff, (1992) Mol Microbiol 6:2419-22; Hu et al., (1987)Cell 48:555-66; Brown et al., (1987) Cell 49:603-12; Figge et al.,(1988) Cell 52:713-22; Deuschle et al., (1989) Proc. Natl. Acad. Sci.USA 86:5400-4; Fuerst et al., (1989) Proc. Natl. Acad. Sci. USA86:2549-53; Deuschle et al., (1990) Science 248:480-3; Gossen, (1993)Ph.D. Thesis, University of Heidelberg; Reines et al., (1993) Proc.Natl. Acad. Sci. USA 90:1917-21; Labow et al., (1990) Mol Cell Biol10:3343-56; Zambretti et al., (1992) Proc. Natl. Acad. Sci. USA89:3952-6; Baim et al., (1991) Proc. Natl. Acad. Sci. USA 88:5072-6;Wyborski et al., (1991) Nucleic Acids Res 19:4647-53; Hillen andWissman, (1989) Topics Mol Struc Biol 10:143-62; Degenkolb et al.,(1991) Antimicrob Agents Chemother 35:1591-5; Kleinschnidt et al.,(1988) Biochemistry 27:1094-104; Bonin, (1993) Ph.D. Thesis, Universityof Heidelberg; Gossen et al., (1992) Proc. Natl. Acad. Sci. USA89:5547-51; Oliva et al., (1992) Antimicrob Agents Chemother 36:913-9;Hlavka et al., (1985) Handbook of Experimental Pharmacology, Vol. 78(Springer-Verlag, Berlin); Gill et al., (1988) Nature 334:721-4.

Active variants or fragments of polynucleotides/polypeptides of interestare also provided. Such active variants can comprise at least 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the native polynucleotide/polypeptide of interest,wherein the active variants retain the biological activity of the nativepolynucleotide/polypeptide.

D. Plants

Plants, plant cells, or seeds having in their genome a genomic windowprovided herein are also encompassed. Plants, plant cells or seedscomprising at least one complex trait locus are also provided. Thegenomic window and complex trait loci of the plants, plant cells orseeds can comprise any combination of any of the various transgenictarget sites, genomic loci of interest or polynucleotides of interestdescribed herein.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which a plant can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included herein, provided that these partscomprise the recited DNA construct.

A transformed plant or transformed plant cell provided herein is one inwhich genetic alteration, such as transformation, has been affected asto a gene of interest, or is a plant or plant cell which is descendedfrom a plant or cell so altered and which comprises the alteration. A“transgene” is a gene that has been introduced into the genome by atransformation procedure. Accordingly, a “transgenic plant” is a plantthat contains a transgene, whether the transgene was introduced intothat particular plant by transformation or by breeding; thus,descendants of an originally-transformed plant are encompassed by thedefinition. A “control” or “control plant” or “control plant cell”provides a reference point for measuring changes in phenotype of thesubject plant or plant cell. A control plant or plant cell may comprise,for example: (a) a wild-type plant or cell, i.e., of the same genotypeas the starting material for the genetic alteration which resulted inthe subject plant or cell; (b) a plant or plant cell of the samegenotype as the starting material but which has been transformed with anull construct (i.e., with a construct which does not express thetransgene, such as a construct comprising a marker gene); (c) a plant orplant cell which is a non-transformed segregant among progeny of asubject plant or plant cell; (d) a plant or plant cell geneticallyidentical to the subject plant or plant cell but which is not exposed toconditions or stimuli that would induce expression of the transgene; or(e) the subject plant or plant cell itself, under conditions in whichthe construct is not expressed.

Plant cells that have been transformed to have any of the variouscomponents provided herein (i.e. transgenic target sites, genomic lociof interest, site-specific recombinases, recombination sites,polynucleotides of interest or any active variants or fragments thereof)can be grown into whole plants. The regeneration, development, andcultivation of plants from single plant protoplast transformants or fromvarious transformed explants is well known in the art. See, for example,McCormick et al. (1986) Plant Cell Reports 5:81-84; Weissbach andWeissbach, In: Methods for Plant Molecular Biology, (Eds.), AcademicPress, Inc. San Diego, Calif., (1988). This regeneration and growthprocess typically includes the steps of selection of transformed cells,culturing those individualized cells through the usual stages ofembryonic development through the rooted plantlet stage. Transgenicembryos and seeds are similarly regenerated. The resulting transgenicrooted shoots are thereafter planted in an appropriate plant growthmedium such as soil. Preferably, the regenerated plants areself-pollinated to provide homozygous transgenic plants. Otherwise,pollen obtained from the regenerated plants is crossed to seed-grownplants of agronomically important lines. Conversely, pollen from plantsof these important lines is used to pollinate regenerated plants. Two ormore generations may be grown to ensure that expression of the desiredphenotypic characteristic is stably maintained and inherited and thenseeds harvested to ensure expression of the desired phenotypiccharacteristic has been achieved. In this manner, the compositionspresented herein provide transformed seed (also referred to as“transgenic seed”) having a polynucleotide provided herein, for example,a transgenic target site, stably incorporated into their genome.

The various components provided herein (i.e. transgenic target sites,genomic loci of interest, site-specific recombinases, recombinationsites, polynucleotides of interest or any active variants or fragmentsthereof) may be used for transformation of any plant species, including,but not limited to, monocots and dicots. Examples of plant species ofinterest include, but are not limited to, corn (maize) (Zea mays),Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly thoseBrassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed include, for example, pines such asloblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine(Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine(Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock(Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoiasempervirens); true first such as silver fir (Abies amabilis) and balsamfir (Abies balsamea); and cedars such as Western red cedar (Thujaplicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). Inspecific embodiments, plants are crop plants (for example, corn,alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut,sorghum, wheat, millet, tobacco, etc.). In other embodiments, corn andsoybean plants are optimal, and in yet other embodiments corn plants areoptimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

It is recognized that the plant having stably incorporated the DNAconstruct can be further characterized for site-specific integrationpotential, agronomic potential, and copy number. See, U.S. Pat. No.6,187,994.

Depending on the polynucleotide(s) of interest incorporated into thegenomic window, the transgenic plants, plant cells, or seeds comprisinga polynucleotide(s) of interest provided herein may have a change inphenotype, including, but not limited to, an altered pathogen or insectdefense mechanism, an increased resistance to one or more herbicides, anincreased ability to withstand stressful environmental conditions, amodified ability to produce starch, a modified level of starchproduction, a altered oil content and/or composition, a alteredcarbohydrate content and/or composition, a altered fatty acid contentand/or composition, a altered phosphorus content and/or composition, aaltered antioxidant content and/or composition, a modified ability toutilize, partition and/or store nitrogen, and the like.

III. Methods of Creating and Altering a Complex Trait Locus

A. Forming a Complex Trait Locus

The components of a genomic window, (i.e. transgenic target sites and/orgenomic loci of interest) can be brought together by various methods.One such method is by crossing plants comprising various target sitesand/or genomic loci of interest having in a given genomic windowdifferent genomic insertion sites and selecting for plants havingundergone a recombination event such that the desired combination oftransgenic target sites and/or genomic loci of interest are present inthe same plant. Such breeding techniques can thereby be employed tocreate a complex trait locus in a plant.

As used herein, “breeding” is the genetic manipulation of livingorganisms. Plants are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinated if pollen fromone flower is transferred to the same or another flower of the sameplant. A plant is sib-pollinated when individuals within the same familyor line are used for pollination. A plant is cross-pollinated if thepollen comes from a flower on a different plant from a different familyor line. In a breeding application, a breeder initially selects andcrosses two or more parental plants. As used herein, “crossing” canrefer to a simple X by Y cross, or the process of backcrossing,depending on the context.

Methods are provided herein to either establish a complex trait locus orto break the complex trait locus apart using breeding techniques. Forexample, a first plant comprising a first transgenic target site withina genomic window, and the first plant does not comprise a first genomiclocus of interest, could be crossed with a second plant comprising thefirst genomic locus of interest within the same genomic window and thesecond plant does not comprise said first transgenic target site withinthe genomic window. A progeny plant is then selected comprising both thefirst transgenic target site and the first genomic locus of interestwithin the genomic window. Selecting a progeny plant comprising both thetarget site and the genomic locus of interest can be done throughvarious methods. For example, a phenotypic analysis can be performedwhereby the activity of a marker or an introduced sequence is detectedin the progeny plant. Alternative methods that assay for markers whichare specific to the genomic locus of interest and the target siteinclude techniques such as PCR, hybridization, Isozyme electrophoresis,Restriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed PCR (AP-PCR), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Amplified Fragment length Polymorphisms (AFLPs), SimpleSequence Repeats (SSRs), and Single Nucleotide Polymorphisms (SNPs).

In non-limiting embodiments, the complex trait locus can comprise (1) atransgenic target site and a genomic locus of interest having differentgenomic insertion sites in said genomic window; (2) 2 transgenic targetsites and a genomic locus of interest having different genomic insertionsites in said genomic window; (3) 2 transgenic target sites and 2genomic loci of interest having different genomic insertion sites insaid genomic window; (4) a genomic locus of interest and a target sitecomprising one or more polynucleotides of interest wherein said genomiclocus of interest and transgenic target site have different genomicinsertion sites; (5) a transgenic target site and a genomic locus ofinterest comprising a transgene, each having a different genomicinsertion site; (6) a transgenic target site and a genomic locus ofinterest comprising a native trait, each having a different genomicinsertion site; (7) a transgenic target site comprising a first and asecond dissimilar recombination sites and a genomic locus of interest,each having a different genomic insertion site; (8) a genomic locus ofinterest, a first transgenic target site comprising a first and a seconddissimilar recombination sites and a second transgenic target sitecomprising a third and a fourth dissimilar recombination sites, whereineach of said genomic locus of interest, first transgenic target site andsecond transgenic target site has a different genomic insertion site;(9) a genomic locus of interest, a first transgenic target sitecomprising a first and a second dissimilar recombination sites, a secondtransgenic target site comprising a third and a fourth dissimilarrecombination sites and a third transgenic target site comprising afifth and a sixth dissimilar recombination sites, wherein each of saidgenomic locus of interest, first transgenic target site, secondtransgenic target site and third transgenic target site has a differentgenomic insertion site; (10) a first transgenic target site and a secondtransgenic target site wherein the second transgenic target sitecomprises different dissimilar recombination sites as the firsttransgenic target site and a genomic locus of interest, each having adifferent genomic insertion site; (11) a first transgenic target site, asecond transgenic target site wherein the second transgenic target sitecomprises the same dissimilar recombination sites as the firsttransgenic target site, and a genomic locus of interest, each having adifferent genomic insertion site; (12) a first transgenic target site, asecond transgenic target site wherein the dissimilar recombination sitescomprise a FRT site or a mutant FRT site, and a genomic locus ofinterest, each having a different genomic insertion site; (13) a firsttransgenic target site and a second transgenic target site wherein thedissimilar recombination sites comprise a FRT5, a FRT6, a FRT7, a FRT12,or a FRT87 site, and a genomic locus of interest, each having adifferent genomic insertion site; or (14) a first transgenic target siteand a second transgenic target site wherein the dissimilar recombinationsites comprise a FRT1 and a FRT87 site, and a genomic locus of interest,each having a different genomic integration site.

A complex trait locus comprising multiple target sites, genomic loci ofinterest and/or polynucleotides of interest can be produced within agenomic window in the genome of a plant. FIG. 1 provides a non-limitingexample of how two traits can be stacked into the genome at a geneticdistance of, for example, 5 cM from each other. A first plant comprisinga first transgenic target site within the genomic window and not havingthe first genomic locus of interest is crossed to a second transgenicplant, comprising a genomic locus of interest at a different genomicinsertion site within the genomic window and the second plant does notcomprise the first transgenic target site. About 5% of the plant progenyfrom this cross will have both the first transgenic target site and thefirst genomic locus of interest integrated at different genomicinsertion sites within the genomic window. Progeny plants having bothsites in the defined genomic window can be further crossed with a thirdtransgenic plant comprising a second transgenic target site and/or asecond genomic locus of interest within the defined genomic window andlacking the first transgenic target site and the first genomic locus ofinterest. Progeny are then selected having the first transgenic targetsite, the first genomic locus of interest and the second genomic locusof interest integrated at different genomic insertion sites within thegenomic window. Such methods can be used to produce a transgenic plantcomprising a complex trait locus having at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more transgenic target sites and/or genomic loci of interestintegrated at different sites within the genomic window. In such amanner, various complex trait loci can be generated.

In one non-limiting embodiment, a method of producing a complex traitlocus in the genome of a plant comprises providing a first plant havingwithin a genomic window of about 10 cM in length at least a firsttransgenic target site and does not comprise a first genomic region ofinterest. The genomic window can be any desired length as describedelsewhere herein. The method involves breeding the first plant to asecond plant which comprises in a different genomic insertion sitewithin the same genomic window a first genomic locus of interest anddoes not comprise the first transgenic target site, and selecting aprogeny plant comprising the first transgenic target site and thegenomic locus of interest. In another embodiment, the method furtherinvolves providing a first plant having within a genomic window a firsttransgenic target site and a second transgenic target site havingdifferent genomic insertion sites wherein the first plant does notcomprise a genomic locus of interest. Breeding the first plant with asecond plant where the second plant comprises a genomic locus ofinterest within the genomic window and does not comprise the first andsecond transgenic target sites, and selecting for a progeny plantcomprising the first transgenic target site, the second transgenictarget site and the genomic locus of interest all having differentgenomic insertion sites within the genomic window. The first transgenictarget site, the second transgenic target site and the genomic locus ofinterest of the progeny plants can segregate independently from oneanother at a rate of about 10-0.1%, about 10-0.5%, about 10-1%, about10-5%, about 9-0.1%, about 9-0.5%, about 9-1%, about 9-5%, about 8-0.1%,about 8-0.5%, about 8-1%, about 8-4%, about 7-0.1%, about 7-0.5%, about7-1%, about 7-4%, about 6-0.1%, about 6-0.5%, about 6-1%, about 6-3%,about 5-0.1%, about 5-0.5%, about 5-1%, about 4-0.1%, about 4-0.5%,about 4-1%, about 3-0.1%, about 3-0.5%, about 3-1%, about 2-0.1%, about2-0.5%, about 1-0.1% or about 1-0.5%.

In this way, it is recognized that the plants provided herein can becrossed to produce a complex trait locus comprising any combination ofthe various genomic windows, transgenic target sites, genomic loci ofinterest, and/or polynucleotides of interest described herein.

B. Altering a Complex Trait Locus

The previous section describes various methods for creating a complextrait locus by adding target sites and/or genomic loci of interest to agenomic window thereby making a complex trait locus. It is recognizedthat a complex trait locus can also be altered by removing orbreeding-away certain transgenic target sites and/or genomic loci ofinterest. The complex trait loci provided herein are designed such thateach transgenic target site and/or genomic locus of interest has adifferent genomic insertion site and can segregate independently. Such adesign allows traits to be bred into the genomic window and also tobreed traits out of the genomic window.

The breeding methods described above for combining traits into a genomicwindow can also be employed to remove traits from a genomic window bybreeding away the trait.

The method of altering a complex trait locus by breeding away comprisesproviding a first plant comprising a transgenic target site and/orgenomic locus of interest to be removed and crossing the first plantwith a second plant that does not have the particular transgenic targetsite and/or genomic locus of interest in the genomic window. Theresulting progeny lacking the transgenic target site and/or genomiclocus of interest would then be selected. For example, a first plantcomprising a first transgenic target site, a second transgenic targetsite and a genomic locus of interest each having a different genomicinsertion site within a genomic window could be crossed to a secondplant. Progeny plants are selected wherein the genomic window does notcomprise any one or any two of said first transgenic target sites orsaid first genomic locus of interest. In this way, at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more target sites and/or genomic loci of interestcan be removed from the complex trait locus.

In one embodiment of the method, the first plant has within a genomicwindow at least a first transgenic target site, a second transgenictarget site, and a first genomic locus of interest. The genomic windowof the first plant is about 10 cM in length and each of the firsttransgenic target site, second transgenic target site and first genomiclocus of interest have different genomic insertion sites and segregateindependently from one another at a rate of about 10% to about 0.1%. Themethod further comprises breeding the first plant to a second plant andselecting progeny wherein the genomic window of the progeny does notcomprise any one or any two of said first transgenic target site, saidsecond transgenic target site, or said first genomic locus of interest.In another embodiment, the genomic window of said first plant is about 5cM in length and the first transgenic target site, the second transgenictarget site and the first genomic locus of interest of the first plantsegregate independently from one another at a rate of about 5% to about0.1%. In a further embodiment, the first transgenic target site or thesecond transgenic target site segregate independently of the firstgenomic locus of interest of the first plant at a rate of about 5% toabout 0.1%.

C. Methods of Altering the Trans genic Target Sites

The transgenic target sites provided herein comprise at least onerecombination site, as described elsewhere herein, which can be utilizedfor direct insertion of one or more polynucleotides of interest into thetarget site. Thus, a complex trait locus comprising various target sitescan be manipulated by site-specific integration methods. Such methodsare described in detail in WO 99/25821, herein incorporated byreference. This method allows removing, adding and/or replacing variouspolynucleotides of interest within transgenic target sites of anestablished complex trait locus by employing site-specificrecombination. Alternatively, the transgenic target site can be alteredin a plant before the plant is utilized in breeding methods to produce acomplex trait locus.

Transgenic target sites can be introduced into the plant genome by anyof the transformation methods known in the art. For example, thetransgenic target site is provided as a polynucleotide construct andintroduced into a plant or plant cell. Then, site-specific integrationcan be employed to insert the transgenic target site into the genome ofa plant. See, for example, U.S. Pat. Nos. 6,187,994, 6,262,341,6,330,545, and 6,331,661 and US Publication Number 2011-0047655, hereinincorporated by reference in their entirety. Once generated, such plantscomprising a transgenic target site can be employed in the breedingmethods discussed above or in a variety of methods to manipulate thesequence within the target site. Such methods employ various componentsof site-specific recombination systems as described in detail elsewhereherein.

The methods also comprise introducing into the plant cell comprising theintegrated transgenic target site a transfer cassette. The transfercassette comprises various components for the incorporation ofpolynucleotides of interest into the transgenic target site within theplant genome. As defined herein, the “transfer cassette” comprises atleast a first recombination site, a polynucleotide of interest, and asecond recombination site, wherein the first and second recombinationsites are dissimilar and correspond to the recombination sites in thetransgenic target site. In some embodiments, the first and secondrecombination sites of the transfer cassette are dissimilar and have adecreased compatibility with respect to one another and correspond tothe recombination sites in the transgenic target site. It is recognizedthat any combination of recombination sites can be employed in thetransfer cassettes to provide a polynucleotide of interest.

In one embodiment, the transfer cassette comprises a first recombinationsite, a first polynucleotide of interest, and a second recombinationsite. In such methods, the first and second recombination sites of thetransfer cassette are recombinogenic (i.e. identical or corresponding)with the first and second recombination sites of the transgenic targetsite, respectively.

In a specific embodiment, the transfer cassette further comprises atleast one coding region operably linked to a promoter that drivesexpression in a plant cell. As discussed elsewhere herein, a recombinaseis provided that recognizes and implements recombination at therecombination sites of the transgenic target site and the transfercassette. The recombinase can be provided by any means known in the artand is described in detail elsewhere herein. In a specific embodiment,the coding region of the transfer cassette encodes a recombinase thatfacilitates recombination between the first and the second recombinationsites of the transfer cassette and the transgenic target site.

Further, the methods provide selecting at least one plant cellcomprising integration of the transfer cassette at the transgenic targetsite. Methods for selecting plant cells with integration at thetransgenic target site, such as selecting for cells expressing aselectable marker, are known in the art and are described elsewhereherein.

As such, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more transgenictarget sites within the complex trait locus can be altered to comprisevarious polynucleotides of interest. Thus, the methods provided hereinhave the benefit of altering a complex trait locus by both breedingmethods and by site-specific integration methods. By such methods, anypolynucleotide of interest can be removed from and/or introduced into acomplex trait locus in a plant.

IV. Methods of Introducing

The methods provided herein comprise introducing into a plant cell,plant or seed various polynucleotide constructs or polypeptidesincluding, but not limited to, the various transgenic target sites,genomic loci of interest, transgenes, a target site comprising a firstand a second dissimilar recombination sites or a first and a seconddissimilar recombination sites having a decreased compatibility withrespect to one another, site-specific recombinases, transfer cassettes,polynucleotides of interest or any active variants or fragments thereofprovided herein.

By “introducing” is intended presenting to the plant the sequence(polypeptide or polynucleotide) in such a manner that the sequence gainsaccess to the interior of a cell of the plant. The methods providedherein do not depend on a particular method for introducing a sequenceinto a plant, only that the polynucleotide or polypeptide gains accessto the interior of at least one cell of the plant. Methods forintroducing sequences into plants are known in the art and include, butare not limited to, stable transformation methods, transienttransformation methods, virus-mediated methods, and sexual breeding.Thus, “introduced” in the context of inserting a polynucleotideconstruct into a cell, means “transfection” or “transformation” or“transduction” and includes reference to the incorporation of apolynucleotide construct into a plant cell where the polynucleotideconstruct may be incorporated into the genome of the cell.

In some embodiments, the plant cells, plants and seeds employed in themethods and compositions have a DNA construct stably incorporated intotheir genome. By “stably incorporated” or “stably introduced” isintended the introduction of a polynucleotide into the plant such thatthe nucleotide sequence integrates into the genome of the plant and iscapable of being inherited by progeny thereof. Any protocol may be usedfor the stable incorporation of the DNA constructs or the variouscomponents employed herein.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct genetransfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballisticparticle acceleration (see, for example, U.S. Pat. Nos. 4,945,050;5,879,918; 5,886,244; and, 5,932,782; Tomes et al. (1995) in Plant Cell,Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology6:923-926); and Lec1 transformation (WO 00/28058). Also see Weissingeret al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987)Particulate Science and Technology 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat.Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein et al. (1988) PlantPhysiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) PlantCell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992)Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant CellReports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

In other embodiments, any of the polynucleotides employed herein may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating a desiredpolynucleotide within a viral DNA or RNA molecule. It is recognized thata sequence employed in the methods or compositions provided herein maybe initially synthesized as part of a viral polyprotein, which later maybe processed by proteolysis in vivo or in vitro to produce the desiredrecombinant protein. Further, it is recognized that promoters employedherein also encompass promoters utilized for transcription by viral RNApolymerases. Methods for introducing polynucleotides into plants andexpressing a protein encoded therein, involving viral DNA or RNAmolecules, are known in the art. See, for example, U.S. Pat. Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al.(1996) Molecular Biotechnology 5:209-221; herein incorporated byreference.

“Transient transformation” is intended to mean that a polynucleotide isintroduced into the host (i.e., a plant) and expressed temporally. Suchtransient transformation methods include, but are not limited to, theintroduction of any of the components (i.e. target sites, genomic lociof interest, recombination sites, site-specific recombinases,polynucleotides of interest or active variants and fragments thereof)directly into the plant or the introduction of the transcript into theplant. Such methods include, for example, microinjection or particlebombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet.202:179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al.(1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) TheJournal of Cell Science 107:775-784, all of which are hereinincorporated by reference. Alternatively, the polynucleotide can betransiently transformed into the plant using techniques known in theart. Such techniques include viral vector system and the precipitationof the polynucleotide in a manner that precludes subsequent release ofthe DNA. Thus, the transcription from the particle-bound DNA can occur,but the frequency with which it is released to become integrated intothe genome is greatly reduced. Such methods include the use particlescoated with polyethylimine (PEI; Sigma #P3143).

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, transformed seed (also referred to as “transgenic seed”)having the recited DNA construct stably incorporated into their genomeis provided.

V. Polynucleotides

Provided herein are polynucleotides comprising the various components ofthe complex trait loci or various components for altering the complextrait loci provided herein (i.e. the various transgenic target sites,genomic loci of interest, transgenes, recombination sites, site-specificrecombinases, transfer cassettes, polynucleotides of interest or anyactive variants or fragments thereof).

The terms “polynucleotide,” “polynucleotide sequence,” “nucleic acidsequence,” and “nucleic acid fragment” are used interchangeably herein.These terms encompass nucleotide sequences and the like. Apolynucleotide may be a polymer of RNA or DNA that is single- ordouble-stranded, that optionally contains synthetic, non-natural oraltered nucleotide bases. A polynucleotide in the form of a polymer ofDNA may be comprised of one or more segments of cDNA, genomic DNA,synthetic DNA, or mixtures thereof. The use of the term “polynucleotide”is not intended to limit the present invention to polynucleotidescomprising DNA. Those of ordinary skill in the art will recognize thatpolynucleotides can comprise ribonucleotides and combinations ofribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides andribonucleotides include both naturally occurring molecules and syntheticanalogues. The polynucleotides provided herein also encompass all formsof sequences including, but not limited to, single-stranded forms,double-stranded forms, hairpins, stem-and-loop structures, and the like.

The compositions provided herein can comprise an isolated orsubstantially purified polynucleotide. An “isolated” or “purified”polynucleotide is substantially or essentially free from components thatnormally accompany or interact with the polynucleotide as found in itsnaturally occurring environment. Thus, an isolated or purifiedpolynucleotide is substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. Optimally, an “isolated” polynucleotide is free ofsequences (optimally protein encoding sequences) that naturally flankthe polynucleotide (i.e., sequences located at the 5′ and 3′ ends of thepolynucleotide) in the genomic DNA of the organism from which thepolynucleotide is derived. For example, in various embodiments, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide is derived.

Further provided are recombinant polynucleotides comprising the varioustarget sites, transgenes, genomic loci of interest, transfer cassettes,recombination sites, site-specific recombinases, polynucleotides ofinterest or any active variants or fragments thereof. The terms“recombinant polynucleotide” and “recombinant DNA construct” are usedinterchangeably herein. A recombinant construct comprises an artificialor heterologous combination of nucleic acid sequences, e.g., regulatoryand coding sequences that are not found together in nature. For example,a transfer cassette can comprise restriction sites and a heterologouspolynucleotide of interest. In other embodiments, a recombinantconstruct may comprise regulatory sequences and coding sequences thatare derived from different sources, or regulatory sequences and codingsequences derived from the same source, but arranged in a mannerdifferent than that found in nature. Such a construct may be used byitself or may be used in conjunction with a vector. If a vector is used,then the choice of vector is dependent upon the method that will be usedto transform host cells as is well known to those skilled in the art.For example, a plasmid vector can be used. The skilled artisan is wellaware of the genetic elements that must be present on the vector inorder to successfully transform, select and propagate host cellscomprising any of the isolated nucleic acid fragments provided herein.The skilled artisan will also recognize that different independenttransformation events will result in different levels and patterns ofexpression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al.,Mol. Gen. Genetics 218:78-86 (1989)), and thus that multiple events mustbe screened in order to obtain lines displaying the desired expressionlevel and pattern. Such screening may be accomplished by Southernanalysis of DNA, Northern analysis of mRNA expression, immunoblottinganalysis of protein expression, or phenotypic analysis, among others.

In specific embodiments, one or more of the polynucleotides describedherein can be provided in an expression cassette for expression in aplant or other organism or cell type of interest. The cassette caninclude 5′ and 3′ regulatory sequences operably linked to apolynucleotide provided herein. “Operably linked” is intended to mean afunctional linkage between two or more elements. For example, anoperable linkage between a polynucleotide of interest and a regulatorysequence (i.e., a promoter) is a functional link that allows forexpression of the polynucleotide of interest. Operably linked elementsmay be contiguous or non-contiguous. When used to refer to the joiningof two protein coding regions, by operably linked is intended that thecoding regions are in the same reading frame. The cassette mayadditionally contain at least one additional gene to be cotransformedinto the organism. Alternatively, the additional gene(s) can be providedon multiple expression cassettes. Such an expression cassette isprovided with a plurality of restriction sites and/or recombinationsites for insertion of a recombinant polynucleotide to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The expression cassette can include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a recombinant polynucleotide provided herein, and atranscriptional and translational termination region (i.e., terminationregion) functional in plants. The regulatory regions (i.e., promoters,transcriptional regulatory regions, and translational terminationregions) and/or a polynucleotide provided herein may be native/analogousto the host cell or to each other. Alternatively, the regulatory regionsand/or a polynucleotide provided herein may be heterologous to the hostcell or to each other. As used herein, “heterologous” in reference to asequence is a sequence that originates from a foreign species, or, iffrom the same species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous polynucleotide isfrom a species different from the species from which the polynucleotidewas derived, or, if from the same/analogous species, one or both aresubstantially modified from their original form and/or genomic locus, orthe promoter is not the native promoter for the operably linkedpolynucleotide. Alternatively, the regulatory regions and/or arecombinant polynucleotide provided herein may be entirely synthetic.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked recombinantpolynucleotide, may be native with the plant host, or may be derivedfrom another source (i.e., foreign or heterologous) to the promoter, therecombinant polynucleotide, the plant host, or any combination thereof.Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation. Toward this end, adapters or linkers may be employed tojoin the DNA fragments or other manipulations may be involved to providefor convenient restriction sites, removal of superfluous DNA, removal ofrestriction sites, or the like. For this purpose, in vitro mutagenesis,primer repair, restriction, annealing, resubstitutions, e.g.,transitions and transversions, may be involved.

A number of promoters can be used in the expression cassettes providedherein. The promoters can be selected based on the desired outcome. Itis recognized that different applications can be enhanced by the use ofdifferent promoters in the expression cassettes to modulate the timing,location and/or level of expression of the polynucleotide of interest.Such expression constructs may also contain, if desired, a promoterregulatory region (e.g., one conferring inducible, constitutive,environmentally- or developmentally-regulated, or cell- ortissue-specific/selective expression), a transcription initiation startsite, a ribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

In some embodiments, an expression cassette provided herein can becombined with constitutive, tissue-preferred, or other promoters forexpression in plants. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, theubiquitin 1 promoter, the Smas promoter, the cinnamyl alcoholdehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, thepEmu promoter, the rubisco promoter, the GRP1-8 promoter and othertranscription initiation regions from various plant genes known to thoseof skill. If low level expression is desired, weak promoter(s) may beused. Weak constitutive promoters include, for example, the corepromoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No.6,072,050), the core 35S CaMV promoter, and the like. Other constitutivepromoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.See also, U.S. Pat. No. 6,177,611, herein incorporated by reference.

Examples of inducible promoters are the Adh1 promoter which is inducibleby hypoxia or cold stress, the Hsp70 promoter which is inducible by heatstress, the PPDK promoter and the pepcarboxylase promoter which are bothinducible by light. Also useful are promoters which are chemicallyinducible, such as the In2-2 promoter which is safener induced (U.S.Pat. No. 5,364,780), the ERE promoter which is estrogen induced, and theAxig1 promoter which is auxin induced and tapetum specific but alsoactive in callus (PCT US01/22169).

Examples of promoters under developmental control include promoters thatinitiate transcription preferentially in certain tissues, such asleaves, roots, fruit, seeds, or flowers. An exemplary promoter is theanther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and 5,689,051).Examples of seed-preferred promoters include, but are not limited to, 27kD gamma zein promoter and waxy promoter, Boronat, A. et al. (1986)Plant Sci. 47:95-102; Reina, M. et al. Nucl. Acids Res. 18(21):6426; andKloesgen, R. B. et al. (1986) Mol. Gen. Genet. 203:237-244. Promotersthat express in the embryo, pericarp, and endosperm are disclosed inU.S. Pat. No. 6,225,529 and PCT publication WO 00/12733. The disclosuresfor each of these are incorporated herein by reference in theirentirety.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced expressionof a polynucleotide of interest within a particular plant tissue.Tissue-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997)Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet.254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168;Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al.(1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) PlantPhysiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505. Such promoters can be modified, ifnecessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590. In addition, the promotersof cab and rubisco can also be used. See, for example, Simpson et al.(1958) EMBO J 4:2723-2729 and Timko et al. (1988) Nature 318:57-58.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed roIC and roID root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene, fused tonptII (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and roIBpromoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See alsoU.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;5,110,732; and 5,023,179. The phaseolin gene (Mural et al. (1983)Science 23:476-482 and Sengopta-Gopalen et al. (1988) PNAS 82:3320-3324.

The expression cassette containing the polynucleotides provided hereincan also comprise a selectable marker gene for the selection oftransformed cells. Selectable marker genes are utilized for theselection of transformed cells or tissues. Marker genes include genesencoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D) and sulfonylureas. Additionalselectable markers include phenotypic markers such as beta-galactosidaseand fluorescent proteins such as green fluorescent protein (GFP) (Su etal. (2004) Biotechnol. Bioeng. 85:610-9 and Fetter et al. (2004) PlantCell 16:215-28), cyan fluorescent protein (CYP) (Bolte et al. (2004) J.Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol.129:913-42), and yellow fluorescent protein (PhiYFP™ from Evrogen; see,Bolte et al. (2004) J. Cell Science 117:943-54). For additionalselectable markers, see generally, Yarranton (1992) Curr. Opin. Biotech.3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol.Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp.177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989)Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg;Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow etal. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc.Natl. Acad. Sci. USA 89:3952-3956; Balm et al. (1991) Proc. Natl. Acad.Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol.10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104;Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill etal. (1988) Nature 334:721-724. Such disclosures are herein incorporatedby reference. The above list of selectable marker genes is not meant tobe limiting. Any selectable marker gene can be used in the compositionspresented herein.

Where appropriate, the sequences employed in the methods andcompositions (i.e., the polynucleotide of interest, the recombinase,etc.) may be optimized for increased expression in the transformedplant. That is, the genes can be synthesized using plant-preferredcodons for improved expression. See, for example, Campbell and Gowri(1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codonusage. Methods are available in the art for synthesizing plant-preferredgenes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, andMurray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporatedby reference.

VI. Libraries Comprising Transgenic Target Sites and Methods of Making

Provided herein is a method of generating a library of plants, seeds orplant cells comprising any of the various transgenic target sitesdescribed herein. The method comprises introducing a recombinantconstruct comprising a transgenic target site into a population ofplants, seeds or plant cells. By “population” is intended a group orcollection of plants, seeds or plant cells. The population can comprise2 or more (i.e. 5, 10, 100, 300, 500, 700, 900, 1100, 1300, 1500, 1700,1900, 2100, 2300, 2500, 2900, 3100, 3300, 3500, 3700, 3900, 4000, 4096,10⁴, 10⁵, 10⁶ or greater) plants, seeds or plant cells. As used herein,a “library” is a population of plants, seeds or plant cells comprisingat least one transgenic target site stably incorporated into theirgenomes.

To generate the plant library, a recombinant construct comprising atransgenic target site is introduced into a population of plant cells.The recombinant construct can be introduced by any means known in theart. The method comprises identifying a plant or plant cell having therecombinant construct and characterizing the genomic insertion site ofthe recombinant construct. A variety of methods are available foridentifying at least one plant cell comprising in its genome atransgenic target site and characterizing the genomic insertion site.Such methods include, but are not limited to, selection based on aselectable marker, PCR methods, sequencing methods, nuclease digestion,Southern blots, and any combination thereof. See, for example, U.S.patent application Ser. No. 12/147,834, herein incorporated by referencein its entirety.

The recombinant construct can integrate at any location in the plantgenome. In one embodiment, the library of plants, seeds or plant cellsis assembled such that each member of the library comprises a targetsite having a different genomic insertion site and when combined into asingle plant genome, can segregate independently from each other. Insuch cases, the integration sites of the transgenic target sites arefound about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10 cM or more from each other in the plant genome.Alternatively, the integration sites of the transgenic target sites arefound about 1-10 cM, about 2-10 cM, about 2-5 cM, about 3-10 cM, about3-6 cM, about 4-10 cM, about 4-7 cM, about 5-10 cM, about 5-8 cM, about6-10 cM, about 6-9 cM, about 7-10 cM, about 8-10 cM, about 9-10 cM,about 0.5-1%, about 0.5-5%, about 0.5-10%, about 0.1-1 cM, about 0.1-2cM, about 0.1-3 cM, about 0.1-4 cM, about 0.1-5 cM, about 0.1-6 cM,about 0.1-7 cM about 0.1-8 cM, about 0.1-9 cM or about 0.1-10 cM fromeach other in the plant genome.

In some embodiments, the library of plants, seeds or plant cellscomprises a population wherein the members of said population have thetransgenic target site at about 10 cM to about 1 cM intervals within adefined genomic window. By “interval” is intended that there is atransgenic target site located at a defined distance from anothertransgenic target site. For example, transgenic target sites located at1 cM intervals within a 10 cM genomic window means that there is atarget site at every 1 cM distance within the genomic window such thatthe genomic window is saturated with target sites. By “saturated” isintended that the library comprises a population of members having thetransgenic target site at about 10 cM intervals to about 0.1 cMintervals, about 10 cM intervals to about 0.5 cM intervals, about 5 cMintervals to about 0.1 cM intervals, about 4 cM intervals to about 0.1cM intervals, about 3 cM intervals to about 0.1 cM intervals, or about 2cM intervals to about 0.1 cM intervals across the entire genomic window.

In some embodiments, the library of plants, seeds or plant cellscomprises a population wherein the members of said population have thetransgenic target site at about 10 cM to about 1 cM intervals within adefined genomic window. The defined genomic window can be any lengthincluding about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60,70, 80, 90, 100, 250, 500, 750, 1000, 2000, 3000, 4000, 5000 cM inlength or up to the complete genome. The genomic window can be about 1-5cM, about 1-10 cM, about 10-30 cM, about 30-60 cM, about 60-100 cM,about 100-500 cM, about 500-1000 cM, about 1000-2500 cM, about 2500-5000cM, or up to the whole length of the genome. In the case that thegenomic window is the entire genome, the plant, seed or plant celllibrary comprises a transgenic target site at every defined interval sothat the entire genome is saturated with transgenic target sites.

Also encompassed herein, is a method for identifying plants or plantcells having a transgenic target site within a given genomic window. Asub-population of plants, seeds or plant cells can be selected from thelibrary such that the transgenic target sites within each member of thelibrary has a different genomic insertion site and segregatesindependently from one another at a rate of about 10% to about 0.1% whenpresent in the same genome.

In another embodiment, the library comprises a population of plants,seeds or plant cells wherein the genomic insertion site of thetransgenic target site in each member of the library segregatesindependently from one another when present in the same genome and themembers of the population have transgenic target sites located at 10 cMintervals to about 1 cM intervals within a genomic window.

These libraries find use in producing a complex trait locus in any givengenomic window by crossing plants within the library or with plants fromlibraries comprising different transgenic target sites. In this way,multiple transgenic target sites can be brought together into a singleplant genome within a given genomic window.

VII. Fragments, Variants and Sequence Comparisons

Active variants and fragments of the various recombination sites,site-specific recombinases and polynucleotides of interest are alsoprovided herein. Biological activity for each of these components isdescribed elsewhere herein.

By “fragment” is intended a portion of the polynucleotide or a portionof the amino acid sequence and hence protein encoded thereby. Fragmentsof a polynucleotide may encode protein fragments that retain thebiological activity of the native protein (i.e., a fragment of arecombinase implements a recombination event). As used herein, a“native” polynucleotide or polypeptide comprises a naturally occurringnucleotide sequence or amino acid sequence, respectively. Thus,fragments of a polynucleotide may range from at least about 20nucleotides, about 50 nucleotides, about 100 nucleotides, and up to thefull-length polynucleotide. A fragment of a polynucleotide that encodesa biologically active portion of a protein employed in the methods orcompositions will encode at least 15, 25, 30, 50, 100, 150, 200, or 250contiguous amino acids, or up to the total number of amino acids presentin a full-length protein. Alternatively, fragments of a polynucleotidethat are useful as a hybridization probe generally do not encodefragment proteins retaining biological activity. Thus, fragments of anucleotide sequence may range from at least about 10, 20, 30, 40, 50,60, 70, 80 nucleotides or up to the full length sequence.

“Variant” sequences have a high degree of sequence similarity. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the native polypeptides. Variants such as these canbe identified with the use of well-known molecular biology techniques,as, for example, with polymerase chain reaction (PCR) and hybridizationtechniques. Variant polynucleotides also include synthetically derivednucleotide sequences, such as those generated, for example, by usingsite-directed mutagenesis but which still encode a protein. Generally,variants of a particular polynucleotide will have at least about 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to that particularpolynucleotide as determined by known sequence alignment programs andparameters.

Variants of a particular polynucleotide (i.e., the reference nucleotidesequence) can also be evaluated by comparison of the percent sequenceidentity between the polypeptide encoded by a variant polynucleotide andthe polypeptide encoded by the reference polynucleotide. Thus, forexample, isolated polynucleotides that encode a polypeptide with a givenpercent sequence identity to a recombinase are known in the art. Percentsequence identity between any two polypeptides can be calculated usingsequence alignment programs and parameters described. Where any givenpair of polynucleotides is evaluated by comparison of the percentsequence identity shared by the two polypeptides they encode, thepercent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

By “variant” protein is intended a protein derived from the nativeprotein by deletion (so-called truncation) or addition of one or moreamino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Variant proteins arebiologically active, that is they continue to possess the desiredbiological activity of the native protein. Such variants may resultfrom, for example, genetic polymorphism or from human manipulation.Biologically active variants of a native protein will have at leastabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the aminoacid sequence for the native protein as determined by known sequencealignment programs and parameters. A biologically active variant of aprotein may differ from that protein by as few as 1-15 amino acidresidues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2,or even 1 amino acid residue.

The following terms are used to describe the sequence relationshipsbetween two or more polypeptides or polynucleotides. As used herein,“reference sequence” is a defined sequence used as a basis for sequencecomparison. A reference sequence may be a portion or the entirety of aspecified sequence. Sequence relationships can be analyzed and describedusing computer-implemented algorithms. The sequence relationship betweentwo or more polynucleotides or two or more polypeptides can bedetermined by generating the best alignment of the sequences, andscoring the matches and the gaps in the alignment, which yields thepercent sequence identity, and the percent sequence similarity.Polynucleotide relationships can also be described based on a comparisonof the polypeptides each encodes. Many programs and algorithms for thecomparison and analysis of sequences are well-known in the art.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 (GCG, Accelrys,San Diego, Calif.) using the following parameters: % identity and %similarity for a nucleotide sequence using a gap creation penalty weightof 50 and a gap length extension penalty weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using a GAP creation penalty weight of 8 and a gap lengthextension penalty of 2, and the BLOSUM62 scoring matrix (Henikoff andHenikoff (1989) Proc. Nall. Acad. Sci USA 89:10915); or any equivalentprogram thereof. By “equivalent program” is intended any sequencecomparison program that, for any two sequences in question, generates analignment having identical nucleotide or amino acid residue matches andan identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find an alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold.

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence. Sequence, particularly polypeptides, that differ byconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated using the selected scoring matrix (BLOSUM62by default for GAP).

Proteins may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants of the recombinase proteins can be prepared bymutations in the DNA. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Kunkel (1985)Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods inEnzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.(1983) Techniques in Molecular Biology (MacMillan Publishing Company,New York) and the references cited therein. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be preferable.

Variant polynucleotides and proteins also encompass sequences andproteins derived from a mutagenic and/or recombinogenic procedure suchas DNA shuffling. With such a procedure, one or more different codingsequences can be manipulated to create a new protein possessing thedesired properties. In this manner, libraries of recombinantpolynucleotides are generated from a population of relatedpolynucleotides comprising sequence regions that have substantialsequence identity and can be homologously recombined in vitro or invivo. Strategies for such DNA shuffling are known in the art. See, forexample, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nat Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

Non-limiting examples of methods and compositions disclosed herein areas follows:

-   1. A plant or seed having in its genome a genomic window comprising    at least a first transgenic target site, a second transgenic target    site, and a genomic locus of interest,

wherein the genomic window is about 10 cM in length;

wherein each of said genomic locus of interest, said first transgenictarget site and said second transgenic target site have differentgenomic insertion sites; and,

wherein each of said first transgenic target site, said secondtransgenic target site, and said genomic locus of interest segregateindependently from one another at a rate of about 10% to about 0.1%.

-   2. The plant or seed of embodiment 1, wherein the genomic window is    about 5 cM in length; wherein each of said first transgenic target    site, said second transgenic target site, and said genomic locus of    interest segregate independently from one another at a rate of about    5% to about 0.1%.-   3. The plant or seed of embodiment 1 or 2, wherein (a) said first    transgenic target site or said second transgenic target site    segregate independently from said genomic locus of interest at a    rate of about 5% to about 0.1%; or, (b) said first transgenic target    site and said second transgenic target site segregate independently    from one another at a rate of about 5% to about 0.1%.-   4. The plant or seed of embodiment 1, 2, or 3, wherein said first    transgenic target site comprises a first recombination site and a    second recombination site, wherein (i) said first and said second    recombination sites are dissimilar with respect to one another;    or (ii) said first and said second recombination sites are    dissimilar and have a decreased compatibility with respect to one    another;-   and said second transgenic target site comprises a third    recombination site and a fourth recombination site, wherein (i) said    third and said fourth recombination sites are dissimilar with    respect to one another; or (ii) said third and said fourth    recombination sites are dissimilar and have a decreased    compatibility with respect to one another.-   5. The plant or seed of any one of embodiments 1-4, wherein said    first transgenic target site or said second transgenic target site    is found within about 5 cM from the genomic locus of interest.-   6. The plant or seed of any one of embodiments 1-4, wherein said    first transgenic target site or said second transgenic target site    is found within about 2 cM from the genomic locus of interest.-   7. The plant or seed of any one of embodiments 1-4, wherein said    first transgenic target site or said second transgenic target site    is found within about 0.5 cM from the genomic locus of interest.-   8. The plant or seed of any one of embodiments 1-7, wherein said    genomic window further comprises a third transgenic target site    comprising a fifth recombination site and a sixth recombination    site, wherein (i) said fifth and said sixth recombination sites are    dissimilar with respect to one another; or (ii) said fifth and said    sixth recombination sites are dissimilar and have a decreased    compatibility with respect to one another; and said third transgenic    target site has a different genomic insertion site than said first    transgenic target site, said second transgenic target site and said    genomic locus of interest.-   9. The plant or seed of embodiment 8, wherein said third transgenic    target site is found within about 5 cM from the genomic locus of    interest.-   10. The plant or seed of any one of embodiments 1-9, wherein said    genomic locus of interest confers a trait comprising male sterility,    site-specific recombination, abiotic stress tolerance, altered    phosphorus, altered antioxidants, altered fatty acids, altered    essential amino acids, altered carbohydrates, herbicide tolerance,    insect resistance or disease resistance.-   11. The plant or seed of any one of embodiments 1-10, wherein said    genomic locus of interest comprises a transgene.-   12. The plant or seed of any one of embodiments 1-10, wherein said    genomic locus of interest comprises a native trait.-   13. The plant or seed of any one of embodiments 1-12, wherein said    first transgenic target site comprises at least one polynucleotide    of interest.-   14. The plant or seed of any one of embodiments 1-12, wherein said    second transgenic target site comprises at least a second    polynucleotide of interest.-   15. The plant or seed of embodiment 4, wherein said second    transgenic target site comprises the same dissimilar recombination    sites as said first transgenic target site.-   16. The plant or seed of embodiment 4, wherein said second    transgenic target site comprises different dissimilar recombination    sites as said first transgenic target site.-   17. The plant or seed of embodiment 4, wherein the dissimilar    recombination sites of said first transgenic target site and said    second transgenic target site comprises a LOX site, a mutant LOX    site, a FRT site or a mutant FRT site.-   18. The plant or seed of embodiment 17, wherein the dissimilar    recombination sites of said first and said second transgenic target    sites comprise a FRT site or a mutant FRT site.-   19. The plant or seed of embodiment 17 or 18, wherein said mutant    FRT site comprises a FRT5 site, a FRT6 site, a FRT7 site, a FRT12    site, or a FRT87 site.-   20. The plant or seed of embodiment 15, wherein the dissimilar    recombination sites of said first and said second transgenic target    sites comprise a FRT1 site and a FRT87 site.-   21. The plant or seed of any one of embodiments 1-20, wherein said    plant or seed is a monocot.-   22. The plant or seed of embodiment 21, wherein said monocot is    maize, wheat, rice, barley, sorghum, or rye.-   23. The plant or seed of any one of embodiments 1-20, wherein said    plant or seed is a dicot.-   24. The plant or seed of embodiment 23, wherein the dicot is    soybean, Bras sica, sunflower, cotton, or alfalfa.-   25. A method for producing a second plant comprising applying plant    breeding techniques to a first plant, or parts thereof, wherein said    first plant is the plant of any one of embodiments 1-24, and wherein    application of said techniques results in the production of said    second plant.-   26. The method of embodiment 25, wherein said second plant comprises    at least one additional transgenic target site or at least one    additional genomic locus of interest within said genomic window when    compared to said first plant; wherein each of said additional    transgenic target site and said additional genomic locus of interest    have a different genomic insertion site with respect to each other    and with respect to said first transgenic target site, said second    transgenic target site and said genomic locus of interest.-   27. The method of embodiment 26, wherein the at least one additional    transgenic target site comprises a polynucleotide of interest.-   28. The method of embodiment 25, wherein said second plant comprises    at least one less transgenic target site or at least one less    genomic locus of interest within said genomic window when compared    to said first plant.-   29. A method of producing a complex trait locus in the genome of a    plant comprising (a) providing a first plant having within a genomic    window at least a first transgenic target site, and wherein said    genomic window is about 10 cM in length and said first plant does    not comprise a first genomic locus of interest; (b) breeding to said    first plant a second plant, wherein said second plant comprises in    said genomic window the first genomic locus of interest and said    second plant does not comprise said first transgenic target site;    and, (c) selecting a progeny plant from step (b) comprising said    first transgenic target site and said genomic locus of interest;    wherein said first transgenic target site and said first genomic    locus of interest have different genomic insertion site in said    progeny plant.-   30. A method of producing a complex trait locus in the genome of a    plant comprising (a) providing a first plant having within a genomic    window at least a first transgenic target site and a second    transgenic target site, wherein said genomic window is about 10 cM    in length, and wherein said first transgenic target site and said    second transgenic target site have a different genomic insertion    site, wherein said first plant does not comprise a first genomic    locus of interest;-   (b) breeding to said first plant a second plant, wherein said second    plant comprises in said genomic window the first genomic locus of    interest, wherein said second plant does not comprise said first    transgenic target site or said second transgenic target site in the    genomic window; and,-   (c) selecting a progeny plant from step (b) comprising said first    transgenic target site, said second transgenic target site and said    first genomic locus of interest; wherein each of said first    transgenic target site, said second transgenic target site and said    first genomic locus of interest have a different genomic insertion    site in said progeny plant; and,

wherein each of said first transgenic target site, said secondtransgenic target site, and said genomic locus of interest in saidprogeny plant segregate independently from one another at a rate ofabout 10% to 0.1%.

-   31. The method of embodiment 30, wherein said genomic window is    about 5 cM in length and wherein each of said first transgenic    target site, said second transgenic target site, and said genomic    locus of interest in said progeny plant segregate independently from    one another at a rate of about 5% to 0.1%.-   32. The method of embodiments 30 or 31, wherein (a) said first    transgenic target site or said second transgenic target site    segregate independently from said first genomic locus of interest at    a rate of about 5% to about 0.1%; or, (b) said first transgenic    target site and said second transgenic target site of said progeny    plant segregate independently from one another at a rate of about 5%    to about 0.1%.-   33. The method of any one of embodiments 30-32, wherein said method    further comprises (a) breeding to said progeny plant a third plant    comprising a second genomic locus of interest, wherein said third    plant comprises in said genomic window said second genomic locus of    interest, wherein said third plant does not comprise said first    transgenic target site, said second transgenic target site or said    first genomic locus of interest in said genomic window; and (b)    selecting a second progeny plant from step (a) comprising said first    transgenic target site, said second transgenic target site, said    first genomic locus of interest, and said second genomic locus of    interest; and-   wherein each of said first transgenic target site, said second    transgenic target site, said first genomic locus of interest and    said second genomic locus of interest have a different genomic    insertion site in said second progeny plant; and,-   wherein each of said first transgenic target site, said second    transgenic target site, said first genomic locus of interest, or    said second genomic locus of interest in said second progeny plant    segregate independently from one another at a rate of about 10% to    about 0.1%.-   34. The method of any one of embodiments 30-33, wherein (a) said    first transgenic target site comprises a first recombination site    and a second recombination site, wherein (i) said first and said    second recombination sites are dissimilar with respect to one    another and, said first transgenic target site comprises a    polynucleotide of interest; or (ii) said first and said second    recombination sites are dissimilar and have a decreased    compatibility with respect to one another and, said first transgenic    target site comprises a polynucleotide of interest; and,-   (b) said second transgenic target site comprises a third    recombination site and a fourth recombination site, wherein (i) said    third and said fourth recombination sites are dissimilar with    respect to one another; and said second transgenic target site    further comprises a second polynucleotide of interest; or-   (ii) said third and said fourth recombination sites are dissimilar    and have a decreased compatibility with respect to one another; and    said second transgenic target site further comprises a second    polynucleotide of interest.-   35. The method of any one of embodiments 30-34, wherein the genomic    position of said first transgenic target site and the first genomic    locus of interest are within 5 cM of each other.-   36. The method of any one of embodiments 30-34, wherein the genomic    position of said first transgenic target site and the first genomic    locus of interest are within 2 cM of each other.-   37. The method of any one of embodiments 30-34, wherein the genomic    position of said first transgenic target site and the genomic locus    of interest are within 0.5 cM of each other.-   38. The method of any one of embodiments 30-31, wherein the first    genomic locus of interest confers a trait comprising male sterility,    site-specific recombination, abiotic stress tolerance, altered    phosphorus, altered antioxidants, altered fatty acids, altered    essential amino acids, altered carbohydrates, herbicide tolerance,    insect resistance or disease resistance.-   39. The method of any one of embodiments 29-38, wherein the first    genomic locus of interest comprises a native trait, a transgene of    interest, or an additional transgenic target site.-   40. The method of embodiment 34, wherein said first transgenic    target site and said second transgenic target site comprise the same    dissimilar recombination sites.-   41. The method of embodiment 34, wherein said first transgenic    target site and said second transgenic target site comprise    different dissimilar recombination sites.-   42. The method of embodiment 34, 40, or 41, wherein the dissimilar    recombination sites comprise a LOX site, a mutant LOX site, a FRT    site or a mutant FRT site.-   43. The method of embodiment 34, 40, or 41, wherein the dissimilar    recombination sites comprise a FRT site or a mutant FRT site.-   44. The method of embodiment 42 or 43, wherein said mutant FRT site    comprises a FRT5 site, a FRT6 site, a FRT7 site, a FRT12 site, or a    FRT87 site.-   45. The method of embodiment 34, wherein said first and said second    transgenic target sites comprise a FRT1 site and a FRT87 site.-   46. A method of altering a complex trait locus in the genome of a    plant comprising (a) providing a first plant having within a genomic    window at least a first transgenic target site, a second transgenic    target site, and a first genomic locus of interest, wherein said    genomic window is about 10 cM in length, and wherein said first    transgenic target site, said second transgenic target site, said    first genomic locus of interest have a different genomic insertion    site;

wherein each of said first transgenic target site, said secondtransgenic target site, or said first genomic locus of interest in saidfirst plant segregate independently from one another at a rate of about10% to about 0.1%;

-   (b) breeding to said first plant a second plant;-   (c) selecting a progeny plant from step (b), wherein said genomic    window from said progeny plant does not comprise any one of or any    two of said first transgenic target site, said second transgenic    target site, or said first genomic locus of interest.-   47. The method of embodiment 46, wherein said genomic window is    about 5 cM in length and wherein each of said first transgenic    target site, said second transgenic target site, or said first    genomic locus of interest in said first plant segregate    independently from one another at a rate of about 5% to about 0.1%;-   48. The method of embodiment 46 or 47, wherein (a) said first    transgenic target site and said second transgenic target site of    said first plant segregate independently from one another at a rate    of about 5% to about 0.1%; or, (b) said first transgenic target site    or said second transgenic target site segregate independently of    said first genomic locus of interest of said first plant at a rate    of about 5% to about 0.1%.-   49. The method of embodiment 46, 47, or 48, wherein-   (a) said first transgenic target site comprises a first    recombination site and a second recombination site, wherein (i) said    first and said second recombination sites are dissimilar with    respect to one another and, said first transgenic target site    comprises a polynucleotide of interest; or (ii) said first and said    second recombination sites are dissimilar and have a decreased    compatibility with respect to one another and, said first transgenic    target site comprises a polynucleotide of interest; and,-   (b) said second transgenic target site comprises a third    recombination site and a fourth recombination site, wherein (i) said    third and said fourth recombination sites are dissimilar with    respect to one another; and said second transgenic target site    further comprises a second polynucleotide of interest; or-   (ii) said third and said fourth recombination sites are dissimilar    and have a decreased compatibility with respect to one another; and    said second transgenic target site further comprises a second    polynucleotide of interest.-   51. The method of any one of embodiments 46-49, wherein the genomic    position of said first transgenic target site and the first genomic    locus of interest are within 2 cM of each other.-   52. The method of any one of embodiments 46-49, wherein the genomic    position of said first transgenic target site and the genomic locus    of interest are within 0.5 cM of each other.-   53. The method of any one of embodiments 46-49, wherein the genomic    locus of interest confers a trait comprising male sterility,    site-specific recombination, abiotic stress tolerance, altered    phosphorus, altered antioxidants, altered fatty acids, altered    essential amino acids, altered carbohydrates, herbicide tolerance,    insect resistance or disease resistance.-   54. The method of any one of embodiments 46-49, wherein the first    genomic locus of interest comprises a native trait, a transgene of    interest, or an additional transgenic target site.-   55. The method of any one of embodiments 25-54, wherein said plant    is a monocot.-   56. The method of embodiment 55, wherein said monocot is maize,    wheat, rice, barley, sorghum, or rye.-   57. The method of any one of embodiments 25-54, wherein said plant    is a dicot.-   58. The method of embodiment 57, wherein the dicot is soybean,    Brassica, sunflower, cotton, or alfalfa.-   59. A method of generating a library of plants, seeds or plant    cells, wherein each of said plant, said seed, or said plant cell in    said library comprise a transgenic target site, said method    comprising: (a) introducing into a population of plant cells a    recombinant construct comprising a transgenic target site; (b)    identifying a plant cell or a plant having the recombinant    construct; (c) characterizing the genomic insertion site of said    recombinant construct within the plant cell or plant of step (b);    and, (d) assembling the library of plants, seeds or plant cells    wherein each member of said library comprises a transgenic target    site having a different genomic insertion site, and, when the    transgenic target sites are combined into a single plant genome,    said transgenic target sites segregate independently from one    another.-   60. The method of embodiment 59, wherein said library of plants,    seeds or plant cells of step (d) comprises a population of plants,    seeds, or plants cells wherein the members of said population have    the transgenic target site at about 10 cM intervals to about 1 cM    intervals within a genomic window.-   61. The method of embodiment 62, wherein the members of said    population have the transgenic target site at about 2 cM intervals    within a genomic window.-   62. The method of embodiment 60 or 61, wherein the genomic window is    the complete genome.-   63. A method for identifying plants or plant cells having a    transgenic target site in a genomic window comprising (a) providing    a library of plants, seeds or plant cells, wherein each of said    plant, said seed, or said plant cell in said library comprises a    transgenic target site in a different genomic insertion site,    wherein the genomic insertion site of said transgenic target site in    each member of said library segregate independently from one another    when present in the same genome; and (b) identifying a    sub-population of plants, seeds or plant cells in said library,    wherein the genomic insertion site of said transgenic target site in    each member of said sub-population segregate independently from one    another at a rate of about 10% to about 0.1% when present in the    same genome.-   64. A library of plants, seeds or plant cells comprising a    population of plants, seeds, or plant cells having a transgenic    target site stably incorporated into their genomes, wherein the    genomic insertion site of said transgenic target site in each member    of said library segregate independently from one another when    present in the same genome and the members of said population have    the transgenic target sites at about 10 cM intervals to about 1 cM    intervals within a genomic window.-   65. The library of plants, seeds or plant cells of embodiment 64,    wherein the genomic window comprises the complete genome.-   66. The library of plants, seeds or plant cells of embodiment 64,    wherein the genomic window is about 10 cM in length.-   67. The library of plants, seeds or plant cells of embodiments 64,    65, or 66, wherein the members of said population have the    transgenic target site at about 2 cM intervals within a genomic    window.-   68. A plant or seed having in its genome a genomic window comprising    at least a first transgenic target site, a second transgenic target    site, and a genomic locus of interest, wherein said genomic    window: (a) is flanked by at least a first marker comprising    UMC1160, UMC2224, NPI579B, PMCB1, IDP3917, GPM199C, IDP1425, MMP68,    UMC2225, STD2C(DBA), TIDP3300, CSU1171, SUT1 or UMC1166, and at    least a second marker comprising AY107207, UMC1568, IDP3783,    BNLG1429, IDP209, LTK1 or IDP7169; or-   (b) is flanked by at least a first marker comprising UMC1625,    UMC2196, UMC2312, BNLG1867, PZA03047, UMC1229, UCK1, RZ390D(CYB5),    MMP20, MMP10, MMP160, PHP20528, UMC2314, UAZ232B(SCI) or UMC2313,    and at least a second marker comprising CDO545, PHP20854, UMC1133,    UFG69, MMP76, Y1, BNLG1422, MMP108B, MMP4, UMC1006, or RZ444E;-   wherein each of said genomic locus of interest, said first    transgenic target site and said second transgenic target site have    different genomic insertion sites; and,-   wherein each of said first transgenic target site, said second    transgenic target site, and said genomic locus of interest segregate    independently from one another at a rate of about 10% to about 0.1%.-   69. Progeny plants obtained from the plant of embodiment 68.-   70. The plant of embodiment 68 further comprising at least one    altered target sequence, wherein the at least one altered target    sequence originated from a corresponding target sequence that was    recognized and cleaved by a double-strand break-inducing agent, and    wherein the at least one altered target sequence is located in said    genomic window.-   71. A method of producing a complex trait locus in the genome of a    plant comprising (a) providing a first plant having within a genomic    window at least a first transgenic target site, wherein said first    plant does not comprise a first genomic locus of interest, and    wherein said genomic window: (i) is flanked by at least a first    marker comprising UMC1160, UMC2224, NPI579B, PMCB1, IDP3917,    GPM199C, IDP1425, MMP68, UMC2225, STD2C(DBA), TIDP3300, CSU1171,    SUT1 or UMC1166, and at least a second marker comprising AY107207,    UMC1568, IDP3783, BNLG1429, IDP209, LTK1 or IDP7169; or-   (ii) is flanked by at least a first marker comprising UMC1625,    UMC2196, UMC2312, BNLG1867, PZA03047, UMC1229, UCK1, RZ390D(CYB5),    MMP20, MMP10, MMP160, PHP20528, UMC2314, UAZ232B(SCI) or UMC2313,    and at least a second marker comprising CDO545, PHP20854, UMC1133,    UFG69, MMP76, Y1, BNLG1422, MMP108B, MMP4, UMC1006, or RZ444E;-   (b) breeding to said first plant a second plant, wherein said second    plant comprises in said genomic window the first genomic locus of    interest and said second plant does not comprise said first    transgenic target site; and, (c) selecting a progeny plant from    step (b) comprising said first transgenic target site and said    genomic locus of interest; wherein said first transgenic target site    and said first genomic locus of interest have different genomic    insertion site in said progeny plant.-   72. A plant or seed having in its genome a genomic window comprising    at least a first transgenic target site, a second transgenic target    site, and a genomic locus of interest, wherein said genomic window:    is flanked by at least a first marker comprising SATT613, SATT284,    S60414-TB or SATT462, and at least a second marker comprising    SATT481, SATT156 or SCT_010;-   wherein each of said genomic locus of interest, said first    transgenic target site and said second transgenic target site have    different genomic insertion sites; and, wherein each of said first    transgenic target site, said second transgenic target site, and said    genomic locus of interest segregate independently from one another    at a rate of about 10% to about 0.1%.-   73. Progeny plants obtained from the plant of embodiment 72.-   74. The plant of embodiment 72 further comprising at least one    altered target sequence, wherein the at least one altered target    sequence originated from a corresponding target sequence that was    recognized and cleaved by a double-strand break-inducing agent, and    wherein the at least one altered target sequence is located in said    genomic window.-   75. A method of producing a complex trait locus in the genome of a    plant comprising (a) providing a first plant having within a genomic    window at least a first transgenic target site, wherein said first    plant does not comprise a first genomic locus of interest, and    wherein said genomic window: is flanked by at least a first marker    comprising SATT613, SATT284, S60414-TB or SATT462, and at least a    second marker comprising SATT481, SATT156 or SCT_010; (b) breeding    to said first plant a second plant, wherein said second plant    comprises in said genomic window the first genomic locus of interest    and said second plant does not comprise said first transgenic target    site; and, (c) selecting a progeny plant from step (b) comprising    said first transgenic target site and said genomic locus of    interest; wherein said first transgenic target site and said first    genomic locus of interest have different genomic insertion site in    said progeny plant.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Production of a Corn Library ComprisingTransgenic Target Sites for Site Specific Integration (SSI)

A method of generating a large collection of transgenic plants or‘library’ of plants, seeds or plant cells comprising any of the varioustransgenic target sites (TTS) for site specific integration (SSI) wasdeveloped for corn as described below.

Development of SSI Platform II Vectors

Vectors for Agrobacterium transformation of corn were developed usingderivative constructs of the original co-integrate Agrobacterium binarysystem described in Komari, et al., 1996. These vectors carried thenecessary molecular elements to enable site specific integration (SSI)based on the FLP/FRT system from yeast. The Agrobacterium constructswith FRT sites to facilitate Recombinase Mediated Cassette Exchange(RMCE) (Seibler and Bode, 1997) were named SSI platform II vectors. Inaddition to the FRT sites, intermediates for SSI platform II vectorsincluded either a multiple cloning site or an Invitrogen™ Gateway®destination site for Gateway® cloning to provide efficient introductionof trait genes for transformation (FIG. 2). Another feature of the SSIplatform II vectors was the inclusion of loxP sites flanking the regionwhere trait genes are introduced to allow cre/lox excision to be usedfor removal of the trait genes if that should be desired. The elementsincluded in SSI platform II vectors allowed for a new gene or multiplegenes to be added to existing genes at a transgenic target site (TTS),including, for example, employing FRT1 and FRT87. If cre/lox excisionwas used in combination with SSI introduction of new genes there was aneffective replacement of existing genes at a transgenic target site. SSIplatform II vectors provided for the placement of value on a transgenictarget site and provided the flexibility to modify the gene content of atransgenic target site considered to be of high value.

A common feature of SSI platform II vectors is the placement of the FRTsites which set up a gene-trapping configuration. The FRT1 site wasplaced between a promoter (for example a maize ubiquitin promoter,UBIZMPRO) as shown in FIG. 2A) and coding region of the maize codonoptimized phosphinothricin acetyltransferase (MO-PAT, PHP35557, FIG. 2A)or phosphomannose isomerase (PMI, PHP44290, FIG. 2B) selectable markergenes. The FRT87 site was placed downstream (three prime) of theterminator sequence of the final selectable marker gene in the SSIregion of SSI platform II vectors (for example a PIN II terminator(PINII TERM) or a Calcium Mosaic Virus 35S terminator, CAMV35S TERM asshown in FIG. 2A). Some vectors comprised a second selectable marker,such as MO-PAT between FRT1 and FRT87 which was driven by a Oryza sativaactin promoter (OS-ACTIN PRO, FIG. 2B). Recombinant insertions wererecovered following introduction of a DNA sequence with a promoterlessselectable marker gene that was not included in the target site andwhich contained the FRT1 site upstream of the coding sequence in thesame way as demonstrated for SSI platform II vectors. The promoterlessmarker constructs, referred to as SSI donor constructs, were introducedby biolistics methods into cells with transgenic target sites. Followingsuccessful RMCE, the initial target site marker (MO-PAT, PMI) was nolonger expressed and the newly introduced marker from the donorconstruct was expressed as a result of the recombination at FRT1 andcapture of the promoter upstream of the gene trap (see U.S. Pat. No.7,102,055). Both selectable and visual markers can be used in the trapto indicate successful site specific integration.

Construct intermediates that include FRT1 and FRT87 sites around theselectable marker gene or genes at the left T-DNA border (LB) region areshown in FIG. 2. Only the T-DNA regions of the constructs are shown forsimplicity. PHP35557 was a derivative of the pSB11 like vector (Komari,et al., 1996) where trait genes are added prior to the formation of thefinal co-integrate construct. PHP44290 is the product of co-integrationbetween pSB1 and pSB11 like intermediates (Komari, et al., 1996) and anInvitrogen™ Gateway® destination site was used in the site for geneintroduction via Gateway® cloning (FIG. 2). An example of one SSIplatform II vector, PHP44556 is shown in FIG. 3. A large number ofrelated constructs were produced with differences in gene numbers, geneactivities, gene sequences, promoters, transcription terminationelements, orientations of genes relative to one another, and positionsof genes between the loxP sites. Each new construct was used to makemultiple corn transformants (n=20 to 100), each transformant with aunique transgenic target site(s). The result of this transformation workbased on SSI platform II was a large number of transgenic target sitesin corn carrying the elements to facilitate site specific integrationand/or trait gene excision and therefore with the capabilities for geneaddition or gene replacement.

Development of a Corn SSI Library

A first population of transgenic plants was generated using a MaizeInbred line 1 (MI1) and involved 16 constructs built from SSI platformII. These constructs were all identical in the T-DNA region to one orthe other of the two constructs shown in FIG. 2 except for the plannedvariation in genes between the loxP sites.

Corn immature embryos were transformed with these constructs by amodified Agrobacterium-mediated transformation procedure, as describedin (Djukanovic et al., 2006). Eight to 10 day embryos were dissectedfrom sterile kernels and placed into liquid medium (4.0 g/I N6 BasalSalts (Sigma C-1416), 1.0 ml/l Eriksson's Vitamin Mix (Sigma E-1511),1.0 mg/l thiamine HCl, 1.5 mg/l, 2, 4 D, 0.690 g/l L-proline, 68.5 g/lsucrose, 36 g/l glucose, pH 5.2). After embryo collection, the mediumwas replaced with 1 ml of Agrobacterium suspension at concentration of0.175-0.45 OD at 550 nm. After incubating for five minutes at roomtemperature, the embryo suspension was poured onto a plate containing4.0 g/l N6 Basal Salts (Sigma C-1416), 1.0 ml/l Eriksson's Vitamin Mix(Sigma E-1511), 1.0 mg/l thiamine HCl, 1.5 mg/l 2, 4 D, 0.690 g/lL-proline, 30.0 g/l sucrose, 0.85 mg/l silver nitrate, 0.1 nMacetosyringone, 3.0 g/l Gelrite, pH5.8). Embryos were incubated in thedark for 3-5 days at 21° C. followed by 3-7 days incubation in the darkat 28° C. on new plates containing 4.0 g/l N6 Basal Salts (SigmaC-1416), 1.0 ml/l Eriksson's Vitamin Mix (Sigma E-1511), 1.0 mg/lthiamine HCl, 1.5 mg/l 2, 4 D, 0.690 g/l L-proline, 30.0 g/l sucrose,0.5 g/l MES, 0.85 mg/l silver nitrate, 100 mg/l carbenicillin, 9.0 g/lagar, pH 5.8). Embryos are then transferred onto new plates containing4.0 g/l N6 Basal Salts (Sigma C-1416), 1.0 ml/l Eriksson's Vitamin Mix(Sigma E-1511), 1.0 mg/l thiamine HCl, 1.5 mg/l 2, 4 D, 0.690 g/lL-proline, 30.0 g/l sucrose, 0.5 g/l MES, 0.85 mg/l silver nitrate, 1.5mg/l bialaphos, 100 mg/l carbenicillin, 6.0 g/l agar, pH 5.8, for 3-4weeks. After 3-4 weeks on the first selection medium, embryos weresub-cultured every 2-4 weeks on 4.0 g/l N6 Basal Salts (Sigma C-1416),1.0 ml/l Eriksson's Vitamin Mix (Sigma E-1511), 1.0 mg/l thiamine HCl,1.5 mg/l 2, 4 D, 0.690 g/l L-proline, 30.0 g/l sucrose, 0.5 g/l MES,0.85 mg/l silver nitrate, 3 mg/l bialaphos for moPAT selection, 100 mg/lcarbenicillin, 6.0 g/l agar, pH 5.8 until transgenic events wereidentified. Regeneration was induced by transferring small sectors oftissue onto maturation media containing 4.3 g/l MS salts (Gibco 11117:Gibco, Grand Island, N.Y.), 5.0 ml/l MS Vitamins Stock Solution, 100mg/l myo-inositol, 0.1 μM ABA, 0.5 mg/l zeatin, 1 mg/l IAA, 60.0 g/lsucrose, 3.0 mg/l bialaphos, 100 mg/l carbenicillin, 6.0 g/l Agar, pH5.6). The plates were incubated in the dark for two weeks at 28° C.Somatic embryos were transferred onto media containing 4.3 g/l MS salts(Gibco 11117: Gibco, Grand Island, N.Y.), 5.0 ml/l MS Vitamins StockSolution, 100 mg/L myo-inositol, 40.0 g/l sucrose, 3.0 mg/l bialaphos,6.0 g/l Agar, pH 5.6, and incubated under artificial light at 28° C. Oneweek later, plantlets were moved into glass tubes containing the samemedium and grown until they were sampled and/or transplanted to soil.

Following exploratory work using maize inbred line 1 and relativelylimited numbers of insertions with available flanking sequence data,another maize inbred line (Maize Inbred line 2) was chosen to continueand extend this work. MI2 was chosen for a combination of superiorgenetics compared to maize inbred line 1 and comparable transformationefficiency. Collection of a larger number of maize inbred line 2transgenic target sites with flanking sequence data was a primary goal.Constructs and molecular methods used to develop this new collection oftransgenic target sites were similar to MI1, but the transformationmethods varied especially in the formulation of the tissue culturemedium. The tissue culture methods for MI2 can be found in Cho, M-J etal., 2011 (US20110165561) and in more detail in Wu, X. E. et al.,(US20100192253). Similar methods were used for both maize inbred linesto recover transgenic plants to the greenhouse, sampling, and downstreamhandling.

Transgenic plants (target lines) were analyzed for T-DNA copy number andfor transgenic target site genomic flanking sequence. Real timequantitative PCR (qPCR) was used to assess the copy number of the T-DNA.A high quality transgenic target site was defined as one that is presentas a single copy in the transgenic plant and one which included no extrasequence from the Agrobacterium vector beyond the T-DNA left or rightborders (RB, LB). One specific, independent qPCR assay was developed foreach gene within the T-DNA borders of each construct. In addition,multiple qPCR assays were used to detect unique sequences of theAgrobacterium constructs outside of the T-DNA borders, often referred toas vector backbone sequence. If all of the genes inside the T-DNA werescored as single copy from the qPCR analysis and there were no vectorbackbone sequences detected, then the transgenic target site wasdescribed as a single copy transgenic target site (single copyinsertion). Those transgenic plants with single copy transgenic targetsites were advanced to the greenhouse and used in further analyses. Eachindependent single copy transgenic target site from the corn SSI librarywas viewed as a unique entity and a potential candidate for sitespecific integration.

Transgenic plants containing single copy T-DNA insertions were sampledas a first or second generation transgenic plant for flanking sequenceanalysis in order to begin further characterization of the transgenictarget site. Multiple leaf samples were taken from each plant and pooledfor DNA extraction. DNA was extracted by using Omega Bioteck E-Z 96Plant DNA Kit following manufacturer's recommendations. Flankingsequence analysis was done either by inverse PCR (IPCR) orligation-mediated nested PCR (LMnPCR) (FIG. 4) followed by sequencing ofthe PCR products. Site specific primers were designed to amplify justinside the T-DNA RB and LB for both of these methods. If IPCR was used,genomic DNA samples were digested with 4-8 different restriction enzymeswhich cut once in the T-DNA sequence near the outside primer bindingsite. The enzymes can cut once at unknown sites in the genomic sequenceoutside of but proximal to the transgenic target site and many othersites in the genome. The DNA fragments with like restriction cuts oneach end were then placed in a ligation reaction to obtain self-ligationto generate small DNA circles consisting of a portion of the T-DNA and asection of genomic DNA. Nested PCR was performed with outward facingprimers designed at each end of the T-DNA sequence of the mini-circles.PCR can amplify a product that contains the ends of the T-DNA sequenceand the adjoining genomic sequence of the transgenic target site(flanking sequence). The small volume of PCR product was treated withExoSAP to clean-up the remaining primers and dNTPS and then the cleanedamplicon was sequenced using the Sanger method. If LMnPCR methods wereused genomic DNA was mechanically sheared into smaller fragments and theends of fragments were converted into blunt ends using Klenow fragmentwith 3′ to 5′ exonuclease activity. Next the fragments were processed bydA-tailing at 3′ ends to prevent blunt-end fragment ligation andprovided a complementary overhang for ligation of the adaptor to thefragments. If the amplicon was sequenced using the Sanger method, thenthe adapter was ligated onto the fragments and nested PCR was performedwith adapter specific primers and primers designed to the T-DNA bordersequences. If the amplicon was sequenced using the Solexa method, thenindexed adapters were ligated to the ends of the DNA fragments forpooled amplicon sequencing and run on a 2100 Bioanalyzer using a DNAspecific chip to check the size and concentration of the product.Sequence results from Solexa were deconvoluted, compared, and evaluatedby using only the best quality reads where confidence was high in theindividual base calls. Finished sequence data was used in BLAST analysisagainst the whole maize genome sequence. This approach resulted inpredictions for which of the 10 corn chromosomes the transgenic targetsite was located on and associated data from that region of thechromosome such as genetic position of the transgenic target sequencefor SSI and markers at that location. Not all of the samples were ableto be resolved in this manner but approximately 70% of total sampleswere able to be assigned to a specific chromosome based on the sequencedata from at least one of the T-DNA borders following first passanalysis (Table 1A and 1B). A subset of the transgenic target sites withdetailed BLAST analysis was predicted to be interrupting endogenousgenes. Transgenic target sites that interrupted endogenous genes wereviewed as undesirable and discarded. Seeds from events comprising theremaining transgenic target sites were kept as seed in a collection thatcould be readily accessed when desired. An early name given to thiscollection was ‘insertion site library’ which referred to the relativelylarge number of samples characterized, the transgenic target sitedistribution which encompassed all 10 maize chromosomes, and thesubsequent seed collection which allowed us to access specific sites inthe maize genome on demand.

TABLE 1A Sample numbers and flanking sequence results for maize inbredline 1. Sample Description (based on flanking sequence results) SampleNo. Single copy insertions submitted for flanking sequence 494 Flankingsequence results obtained 477 Flanking sequence assigned to a chromosome329 Transgenic target sites predicted to be interrupting an 172endogenous gene Samples with both T-DNA borders resolved 405 Sampleswith both T-DNA borders in repetitive sequence 46

TABLE 1B Sample numbers and flanking sequence results for maize inbredline 2. Sample Description Sample No. Quality insertions submitted forflanking sequence 1286 Flanking sequence results obtained 1157 Flankingsequence assigned to a chromosome 810 Transgenic target sites predictedto be interrupting an 420 endogenous gene Samples with both T-DNAborders resolved 347 Samples with both T-DNA borders in repetitivesequence 81

Obtaining flanking sequence data for single copy transgenic target sitesand being able to get back to the transgenic material through the seedcollection are the first steps in the process toward advancing them intosite specific integration work and building complex trait loci. Theinitial flanking sequence data was generated for a large number oftransgenic plants by relatively rapid screening. When one of the T-DNAborder regions did not resolve or there was a conflict between resultsfor the RB and LB, the plant was still maintained for the collection. Ifno flanking sequence (FS) results were obtained for a plant, then thatplant was discarded from the collection. When an transgenic target sitefrom the library was identified that was in a region of interest (alsoreferred to as a genomic locus of interest) in the maize genome then theseed of that plant was grown out and samples were re-submitted forflanking sequence analysis. Once a confirmation of the original flankingsequence was obtained, additional work including Southern analysis toverify qPCR results and SSI transformation work to characterizefrequency for the site was initiated. If a transgenic target site forSSI passed Southern analysis to confirm that there was a single intactcopy of the T-DNA and no vector backbone, and it was demonstrated toundergo SSI, then it became a recombinant target locus (RTL) and wasassigned a unique identifier. RTLs were advanced further forcharacterization of gene expression and agronomics. Currently more than1000 transgenic target sites have been identified spanning all of themaize chromosomes.

Example 2 Production of a Complex Trait Locus (CTL3A) at Chromosome 1

One valuable region of the corn genome, referred to as Complex TraitLocus 3A (CTL3A; FIG. 5a ), was identified containing a trait ofinterest (Trait 3A) on chromosome 1 as follows.

Identification of Trait of Interest

The location of Trait3A on chromosome 1 was determined based on sequencehomology following determination of the transgenic target site flankingsequence and then verified by mapping data. Transgenic target siteflanking sequence (FS) was obtained by using inverse PCR (IPCR) andamplified DNA products were sequenced using the Sanger sequencingmethod. Once sequence was obtained, the results were compared to themaize whole genome sequence using the BLAST algorithm to match FS datato maize genome sequence. The maize genome sequence was assembled intosubset sequences for each individual chromosome so that BLAST analysiscould be used to predict the location of the FS to a chromosome. Themaize genetic map was associated with the chromosome sequence based onDNA marker sets and this allowed a position on the chromosome to beidentified for a particular FS. Finally, the chromosome and position ofthe Trait3A insertion that was initially determined usingbio-informatics was verified by mapping the transgenic target site.

Identification of Transgenic Events Comprising Transgenic Target Sites(TTS) Located Near the Trait of Interest

In order to develop a Complex Trait Locus at the Trait3A location, oneor more independent transgenic loci in close genetic proximity of Trait3A on the corn genome needed to be generated. These independenttransgenic loci can then be combined by breeding or re-transformationand can segregate in subsequent breeding steps as a single genetic locus(as described in FIG. 1).

A set of 8 SSI platform II constructs was used to generate maize inbredline 1 (MI1) transgenic plants (containing independent transgenic loci)as described in Example 1 (Table 1A). There were 50 to 100 transgenicplants with single copy T-DNA insertions generated for each of the 8constructs. Leaf samples from most of these transgenic plants weresubmitted for flanking sequence analysis and the final set of resultsincluded nearly 500 transgenic individuals. Approximately 67% (329/494)of the transgenic plants submitted gave sequence results that could beassociated with one of the ten maize chromosomes followingbio-informatics analysis involving the BLAST algorithm (data not shown).Those that could not be associated to a maize chromosome were eitherfailed samples of the flanking sequence method or samples with sequenceresults included only in repetitive genome sequence. Each constructprovided multiple transgenic target sites for SSI that were distributedacross each of the 10 corn chromosomes.

FIG. 5A shows transgenic target sites identified on Chromosome 1 neartwo transgenic traits of interest, Trait3A and Trait3C. Each individual(single border) or pair (both borders) of triangles and bars across thechromosome indicates unique transgenic target sites. FIG. 5A shows 15transgenic target sites mapped to chromosome 1. The transgenic targetsites of Trait3A and Trait3C were mapped to chromosome 1 using primarilya bio-informatics approach. Using flanking sequence results from ourlibrary of approximately 500 SSI platform II insertions in maize inbred1 (individual transgenic events), 4 independent transgenic target sitesfor site specific integration (TTS-3A1, TTS-3A2, TTS-3A3, TTS-3A4) wereidentified that could be located very near (plus or minus approximately5 centi-Morgans) to the Trait3A locus (also referred to as the genomiclocus of interest) and together could comprise the Complex Trait Locus3A (CTL3A) if properties of each transgenic target site warrantedfurther development. In addition, an insertion site (IS) (IS-3A5, alsoreferred to as MHP14, see U.S. Provisional Patent Application No.61/466,602, filed on Mar. 23, 2011) was developed in this region usinghoming endonuclease technology and recombination. Each of these TTSs orIS (TTS3A1-TTS3A4 and IS3A5) can be used independently or in combinationwith Trait3A and physically linked by crossing with Trait3A. Theresulting complex trait locus can be introduced into a breeding programacting as a single genetic locus.

The 4 transgenic events representing the 4 transgenic target sitessurrounding Trait3A were selected for follow up work includingadditional characterization undertaken prior to complex trait locusformation. Seed was not available for the transgenic lines representingTTS-3A1, while transgenic lines representing TTS-3A3 and TTS-3A4 werepredicted to interrupt endogenous genes and were therefore undesirablefor complex trait locus development and therefore abandoned. Thetransgenic line comprising TTS-3A2 (also referred to as insertion site98281928) was generated using an Agrobacterium vector derived from SSIplatform II intermediate that was closely related to PHP35557 (FIG. 2)except that it incorporated the Invitrogen™ Gateway® destination sitefor Gateway® cloning.

Table 2 shows the public IBM2 genetic map position (IBM genetic map dataavailable through the MaizeGDB website) as well as the map position ofan internally derived single meiosis map (PHB) for a multitude ofmarkers, transgenic target sites and insertion sites (TTSs and IShighlighted in gray). PHB is a genetic map based on a population thathas undergone one round of meioses (e.g. an F₂) whereas the IBM2 mapconsist of multiple meioses. TTS-3A6 is also referred to as insertionsite 148053664, TTS-3A7 is also referred to as insertion site 152323453,TTS-3A8 is also referred to as insertion site 154587278, TTS-3A9 is alsoreferred to as insertion site 153175440, and TTS-3A10 is also referredto as insertion site 148016489.

Table 2 shows that the Trait3A insertion resides at IBM position 134.66in bin 1.02. Transgenic target site TTS-3A2 and insertion site IS-3A5were identified from MI1 and an additional 5 TTS sites (TTS-3A6,TTS-3A7, TTS-3A8, TTS-3A9, TTS-3A10) near the Trait3A genomic locus ofinterest were identified from our MI2 collection. One of the eventscomprising TTS-3A7 (Table 2) was advanced for further characterization.

TABLE 2 Complex Trait Locus CTL-3A on Chromosome 1 of corn. IBM PHBLocus Position position UMC1160 108.3 44.1 UMC2224 110.9 45.2 NPI579B112.2 45.9 PMCB1 115.2 46.6 TTS-3A2 116.2 49.6 TTS-3A6 116.9 50.4IDP3917 117.0 GPM199C 117.2 TTS-3A7 119.5 51.0 TTS-3A8 119.5 51.0IDP1425 119.7 MMP68 123.6 51.5 UMC2225 124.7 52.0 STD2C(DBA) 125.3IS-3A5 125.3 52.1 TIDP3300 125.4 CSU1171 127.3 53.2 SUT1 133.6 UMC1166133.6 TRAIT3A 138.6 55.0 AY107207 139.4 UMC1568 141.8 54.1 IDP3783 141.8TTS-3A9 142.1 58.0 TTS-3A10 142.5 59.1 BNLG1429 143.5 57.8 IDP209 146.058.1 LTK1 148.0 58.6 IDP7169 153.2 63.3

FIG. 6 shows a schematic of the transgenic target sites of the CTL3Acomplex trait locus in relation to the genomic window of interest(TRAIT3A) and public BACS on the maize physical map.

Site Specific Integration in Transgenic Maize Event Comprising TTS-3A2

The transgenic event representing TTS-3A2 (Table 2 and FIG. 6) wasfurther evaluated by testing its capability for site specificintegration of genes of interest. Each FRT site of the event (FRT1 andFRT87), and some DNA sequence downstream and upstream of the transgenictarget site were amplified by PCR using oligonucleotide primershomologous to sequence of the T-DNA directly adjacent to the FRT siteson both sides. The PCR products were purified by agarose gelelectrophoresis. A band with the expected size of the predicted PCRproduct was cut from the gel, extracted, submitted for DNA sequencingand the FRT target site sequence was confirmed to be intact. This dataconfirmed that SSI with TTS-3A2 would not be impeded by the FRT sitessince they were intact and demonstrated the expected sequence result.

Particle bombardment was used as the method of DNA delivery for SSItransformation. A ‘donor plasmid’ containing a sequence of interestflanked by FRT sites was delivered into heterozygous immature embryoscontaining TTS-3A2 via biolistic-mediated transformation. Nine to 11day-old immature embryos (1-1.5 mm in size) were dissected fromsterilized kernels and plated with their axis down to media containing4.0 g/l N6 Basal salts (Sigma C-1416) 1.0 ml/l Eriksson's Vitamin Mix(Sigma E-1511). 1.0 mg/l thiamine HCl, 1.5 mg/l 2,4-D, 0.690 g/lL-proline, 30 g/l Sucrose, 0.85 mg/l silver nitrate, 3.0 g/l Gelrite, pH5.8 and incubated in the dark at 28° C. for 3 to 5 days beforeintroduction of DNA. Two to four hours prior to bombardment the embryoswere plasmolized by placing them on the above media containing 120 gm ofsucrose.

Plasmid DNA was associated with the gold particles by mixing 50 ng ofPHP27064, 10 ng of PHP5096 (UBI:FLPm), 25 ng of the helper plasmidPHP31729 (OLE PRO:ODP2), and 25 ng of the helper plasmid PHP21139 (IN2PRO:WUS) (volume of the DNA solution was adjusted to 40 μl), 50 μl of1-μm gold particles at 0.01 mg/μl and 5 μl TFX-50 (Promega E1811/2)(FIG. 7). SSI could be completed using only PHP27064 and PHP5096, butearlier experiments (data not shown) showed that inclusion of plasmidswith the genes on PHP21139 and PHP31729 increased SSI frequency. Theseadditional genes help to stimulate cell division in culture and mayenhance recombination (see U.S. Patent Publication No. US20110165679A1).The particle/plasmid solution was allowed to gently mix for 10 minutes.The particles and attach DNA were then spun down for 1 minute at 10,000rpm, the supernatant removed and replaced with 120 μl of 100% ethanol.The particles were then re-suspended by gentle sonication, 10 μl of theparticle solution was spotted on each carrier disc and the EtOH wasallowed to evaporate. The macro carrier was placed 2.5 cm from a 450 psirupture disc with the immature embryos placed on a shelf 7.5 cm belowthe launch assembly.

After bombardment the embryos were removed from the high sucrose mediaand placed back on same medium containing 30 g/l sucrose. The embryoswere incubated in the dark at 28° C. for 7 days at which time theembryos were moved to selection plates of the above media containing0.1778 mg/l glyphosate. Embryos were subcultured to fresh medium after 3weeks; transgenic events were identified 4 weeks later. Transgenicevents growing under selection were then observed for their yellowfluorescent protein positive phenotype. Those that exhibited afluorescent phenotype indicative of RMCE were regenerated usingglyphosate as the selective agent at the same levels presented above.Plantlets were sampled and/or transplanted to soil.

The expected result is shown in FIG. 8 as Target with RMCE.

A series of qPCR assays were used to indicate that SSI had occurredincluding checking the copy number of each gene between the FRT sitesthat originally resided in the target and donor plasmids as well asdetection assays for the other co-bombarded plasmids. In order to obtainthese results DNA was extracted from leaf samples of regenerated plantsvia a modified alkaline lyses method using 1 punch (200 ng) of freshleaf tissue (Truett et al., 2000). For real-time quantitative PCR(qPCR), each gene was quantitated using proprietary forward and reverseprimers along with a corresponding FAM based probe. Each assay wasprimer titrated and normalized to an amplification signal from a singlecopy native sequence which was detected by a second distinct primer setand VIC-based probe. Each amplification reaction for determination ofcopy number of the HT (Herbicide tolerance), YFP, and MO-PAT genes wererun simultaneously with the normalizing gene in a single tube reaction.Upon completion of the qPCR, all raw data were used to calculate the dCTvalues. Copy number determination was computed with the ΔΔCT method asdescribed in the ABI (Applied Biosystems, Foster City, Calif.) userbulletin #2. Endpoint positive and negative qPCR calls were made forFLP, ODP2, WUS, and the two recombinant junction assays (UBI-FRT1-HT,donor-FRT87-target) according to the dCTs estimations. A qPCR reactionrunning 5 cycles later than the native normalizing sequence was callednegative.

There were approximately 1400 immature embryos heterozygous for Insert 1that were co-bombarded using the donor plasmid, PHP27064, and the SSIassociated plasmids with FLP recombinase, ODP2, and WUS (FIG. 7).Bombarded embryos were transferred to selective culture medium asdescribed above and then cultured for callus growth. Callus growing inthe presence of glyphosate and showing a yellow fluorescent phenotypewhen viewed under a dissecting microscope with the appropriate lightsource and filters was advanced for plant regeneration. Two independentcalli with these properties were advanced into regeneration, regeneratedplants were recovered, and leaf samples from the plants submitted forqPCR. The set of qPCR assays run included assays that span the FRT1 andFRT87 recombinase sites and include primers designed to both target(Insertion 2) and donor sequence to yield the result (Table 3). Apositive (POS) call for FRT1 and FRT87 indicate that SSI has occurred.Additional assays were run to assess the quality of the SSI. Copy numberassays were run for the genes in the donor, HT and YFP, to determinewhether additional copies of the donor plasmid were randomly inserted.Assays for ODP2, WUS, and FLP were run for the same reason and the bestresults were negative for ODP2 and FLP and 1 copy for WUS (assay detectsendogenous WUS gene). The last assay was a detection method for the genein the target sequence, PAT, which should have been removed as a resultof site specific integration. The cumulative results of this analysisshowed that SSI had occurred in both recovered calli and thatundesirable integration of the co-bombarded plasmids was unlikely (Table3). Southern analysis can be used for the further confirmation, but theqPCR analysis is a valuable initial screen. The SSI frequency obtainedfor TTS-3A2 was 0.14% based on the total embryo number.

TABLE 3 qPCR analysis of regenerated plants from two glyphosateresistant, yellow fluorescent calli from the event comprising TTS-3A2.This analysis was used to detect SSI and the presence of plasmidsincluding PHP21139, PHP5096, PHP31729, and PHP27064. Callus Plant YFP HTYFP No. No. Pheno. FRT1 FRT87 COPY COPY PAT ODP2 WUS FLP 1 1 POS POS POS1 1 NEG NEG 1 NEG 1 2 POS POS POS 1 1 NEG NEG 1 NEG 1 3 POS POS POS 1 1NEG NEG 1 NEG 1 4 POS POS POS 1 1 NEG NEG 1 NEG 1 5 POS POS POS 1 1 NEGNEG 1 NEG 1 6 POS POS POS 1 1 NEG NEG 1 NEG 2 1 POS POS POS 0.5 0.5 NEGNEG 1 NEG 2 2 POS POS POS 0.5 0.5 NEG NEG 1 NEG 2 3 POS POS POS 0.7 0.6NEG NEG 1 NEG POS = Positive. NEG = Negative. YFP = yellow fluorescentprotein (ZS-Yellow1 NI, Clontech). HT = Herbicide tolerance gene. ODP =ovule development protein. WUS = wuschel. FLP = flp recombinase.

In summary of the work above, a transgenic event containing TTS-3A2 wasgenerated using a construct derived from the SSI platform II vectorintermediate and referred to as IE-2. The transgenic target site wascharacterized as a quality insertion using qPCR analysis during thetransformation process. Leaf samples from IE-2 were submitted forflanking sequence analysis. The resulting flanking sequence data wasused to estimate its genomic position very near to Trait3A onchromosome 1. Later mapping data confirmed the chromosome 1 locationinitially estimated using flanking sequence data. IE-2 was furthercharacterized for use in SSI transformation experiments by confirmationof intact FRT site sequence. Next, the process of SSI transformation wasinitiated to determine a frequency of obtaining recombinase mediatedcassette exchange (RMCE) from IE-2. Transgenic plants showing thecorrect phenotype for SSI were recovered from these experiments. SSI wasverified by using multiple qPCR assays which showed replacement of themopat gene by the cassette with the HT and YFP genes and no integrationof the intact plasmids that had been co-bombarded with the donorplasmid.

Generating a Transgenic Plant Containing the Complex Trait Loci 3A

In order to build the complex trait locus 3A, additional trait genes canbe inserted into TTS-3A2 of IE-2. The resulting transgenic plantcontaining the additional trait genes in TTS-3A2 can then be crossed (orre-transformed) with transgenic events containing the Trait3A (asillustrated in FIG. 1). The addition of trait genes at TTS-3A2 can beaccomplished by developing a donor plasmid with MO-PAT in the gene trapposition and trait genes downstream of the marker gene and thenrepeating the transformation protocol detailed above except thatbialaphos or phophinothricin would replace glyphosate as the selectionagent in tissue culture and qPCR assays would match the mo-pat gene.

TTS-3A2 was predicted to be 3.6 cM away from Trait3A (Table 2) based onan internally derived single meiosis genetic map (PHB), indicating a100% chance of finding at least 1 recombinant in 400 progeny. Crosseshave been made to link TTS-3A2 with Trait3A.

Related work with another transgenic target site in the same region andlocated closer to Trait3A was completed. This insertion site IS-3A5(also referred to as MHP14), was generated by meganuclease assistedhomologous recombination (described in U.S. Provisional PatentApplication No. 61/466,602, filed on Mar. 23, 2011) (FIG. 5A, Table 2).The site was predicted to be less than 2 cM away from Trait3A based onan internally derived single meiosis genetic map (PHB) (Table 2). Thebreeding scheme for physical linkage should work for any 2 insertionsites or genomic points of interest (transgenic insertions, nativetraits, QTL's, haplotypes, chromosomal regions of interest, etc.), orcombinations of the two (Table 4). Briefly, an F1 plant is created bycross pollination of two plants homozygous for the entities that need tobe linked. F1 kernels are germinated and screened by qPCR or anothermolecular method to verify that both are present in the F1. When the F1plant is flowering, then pollen is carried from the F1 to anon-transgenic recipient plant or a line that does not carry theentities to be linked. Progeny from that cross are screened forindividuals that carry both entities and they should be linked at thatpoint from recombination that took place during pollen formation of theF1 plant. The individuals identified to have both entities are thenself-pollinated and the progeny will segregate 1:2:1 (1 homozygous:2heterozygous:1 null) for the two entities together. The 1:2:1segregation ratio at this final breeding step confirms that the twoentities are physically linked at a single genetic locus.

TABLE 4 Breeding scheme for selection of linked transgenic target sitesor other entities and confirmation of linkage. GEN. Materials GeneticStatus Breeding Step Result 1 Entity 1, Entity 2 Homozygous for Entity 1× Entity 2 F1 (1, 2) one locus each not linked 2 F1, Entity free F1heterozygous Entity Free × F1 F2 (1, 2, 1:2, null) line for both loci(female × male) (1:2 linked) 3 F2 Heterozygous F2 (1:2) Self F3 (1:2,null) linked screen. pollinated 4 F3 Single locus None 1:2:1 (1:2, null)entity 1:entity 2 (homo:hemi:null) GEN. = Plant generation.

Seed comprising the IS-3A5 and Trait3A were planted in subsequent weeklyplantings and the plants matured to flowering stage. At flowering,plants with IS-3A5 were used as a pollen source and crossed with thetransgenic plants containing the Trait3A, thereby creating a CTL3Acomplex trait locus.

Ears were harvested from these crosses, dried down, and F1 kernelsremoved. The F1 kernels were planted and the resulting plants werescreened as seedlings by qPCR to confirm that both transgenic loci werepresent. At the same time the IS-3A5/Trait3A kernels were planted,kernels of another suitable non-transgenic line were planted to act as afemale in crosses with the F1 transgenic line. As these plants flowered,pollen from the IS-3A5/Trait3A transgenic F1 was carried to ears of thenon-transgenic line. Filled ears from these crosses were processed andresulting F2 kernels planted in larger numbers in flats to screen forrecombinants between IS-3A5 and Trait3A. This screening was based onqPCR analysis with two assays specific to Trait3A and one assay specificto IS-3A5. In total, 813 F2 plants were screened resulting in 419positive for CTL3A only, 387 positive for IS-3A5 only, and 7 plants thatwere positive for both transgenic loci. Assuming the 7 double positivewere all recombinants as expected, the frequency is 0.9% indicating thatIS-3A5 is approximately 1 cM away from CTL3A. These 7 plants were grownup and self-pollinated and the rest of the F2 progeny discarded.Confirmation of the linkage of IS-3A5 and Trait3A in these 7 plantsincluded analysis of the progeny from the self-pollination. Bothtransgenic loci would segregate 1 homozygous:2 heterozygous:1 null ifthey were linked at one locus. Leaf samples from progeny of one of theseven self-pollinated F2 plants were taken for qPCR analysis. Two of theqPCR assays, MHP14 HR1 and CTL3A, were designed for detection only andnot to indicate copy numbers of the amplicon sequences. Two additionalqPCR assays, MHP14 IS and CTL3A gene 3, were developed to indicate thecopy numbers of the amplicon (Table 5). The transgenic insertion at theIS-3A5 target site excludes detection by the qPCR assay. If theinsertion was not present, the qPCR was positive and the locus isdesignated as wild type (wt). The MHP14 IS assay is quantitative and aresult with 2 wt alleles indicated that the IS-3A5 transgenic insertionwas not present. The Trait3A GENE3 assay was designed in the openreading frame sequence of one of the genes in the CTL3A stack. It is aquantitative assay such that a 2 copy call indicated that the donorplant was homozygous for Trait3A and a 1 copy call is heterozygous. Theother two assays, MHP14 HR1 and Trait3A, did not indicate the number ofinsertion copies but supported the copy number calls withpositive/negative indications for related elements. For example, ahomozygous plant for both transgenic alleles would have 0 wt copies ofthe MHP14 IS, be positive for the presence of MHP14 HR1, contain twocopies of Trait3A GENE3, and would be positive for the distinctadditional assay for the CTL3A insertion (Table 5). In conclusion, theanalysis demonstrated that there were 8 homozygous:16 heterozygous:7null and this matched the expected result for two transgenes linked at asingle genetic locus. This confirmed our ability to link the two lociinto a single trait locus on chromosome 1 and create a Complex traitLoci 3A.

TABLE 5 qPCR analysis of progeny from a self-pollination of a single F2plant with two transgenic loci, IS-3A5 (MHP14) and CTL3A. Number MHP14CTL3A Plants MHP14 IS IS CALL MHP14 HR1 GENE 3 CTL3A 8 0 No wt allelespositive 2 copy positive 16 1 1 wt allele positive 1 copy positive 7 2 2wt alleles negative negative negative

Example 3 Production of a Complex Trait Locus (CTL6A) on Chromosome VI

Another valuable region of the corn genome, referred to as Complex TraitLocus 6A (CTL6A; FIG. 5b ), was identified containing a trait ofinterest (Trait 6A) on chromosome VI as follows.

Identification of Trait of Interest

The genetic map position of Trait6A on chromosome VI was determinedbased exclusively by trait mapping data obtained during the process ofinbred conversions and trait integration.

Identification of Transgenic Events Comprising SSI Target Sites LocatedNear the Trait of Interest

In order to develop a Complex Trait Locus at the Trait6A location, oneor more independent transgenic loci in close genetic proximity ofTrait6A on the corn genome needed to be generated. These independenttransgenic loci can then be combined by breeding or re-transformationand can segregate in subsequent breeding steps as a single genetic locus(as described in FIG. 1).

Initial screening of the flanking sequence data for transgenicinsertions from maize inbred line 1 (MI1) resulted in the identificationof four transgenic target sites (TTS-6A1, TTS-6A2, TTS-6A3, TTS-6A4)near Trait 6A (FIG. 5B). Bars across the chromosome indicate uniqueinsertion sites and represent potential members of the Complex TraitLocus 6A (CTL6A). Each of these 4 TTSs can be used independently or incombination with Trait6A and physically linked by crossing with Trait6A.Seed was obtained from a transgenic event containing the TTS-6A3 site(FIG. 5B, arrows), referred to as IE-7, and a transgenic eventcontaining TTS-6A4 (FIG. 5B, arrows), referred to as IE-8. IE-8 wasdropped due to the presence of extra bands in Southern analysis. TTS-6A3is also referred to as 97757511 and TTS-6A4 is also referred to as97757502.

The genetic map position was used in combination with the physical mapto identify MI2 insertion sites that were predicted to be within 5 cM oneither side of Trait6A (FIG. 9). The maize genetic map facilitated theassociation with the chromosome sequence and DNA marker sets

Table 6A shows the public IBM2 genetic map position (IBM genetic mapdata available through the MaizeGDB website) as well as the map positionof an internally derived single meiosis map (PHB) for a multitude ofmarkers and transgenic target sites (insertion sites; TTSs highlightedin gray). PHB is a genetic map based on a population that has undergoneone round of meioses (e.g. an F₂) whereas the IBM2 map consist ofmultiple meioses. TTS-6A5 is also referred to as insertion site145401580, TTS-6A6 is also referred to as insertion site 124537396,TTS-6A7 is also referred to as insertion site 148174073, TTS-6A8 is alsoreferred to as insertion site 145401461, TTS-6A9 is also referred to asinsertion site 148304686, TTS-6A10 is also referred to as insertion site147136301, TTS-6A11 is also referred to as insertion site 145403827,TTS-6A12 is also referred to as insertion site 145403004, TTS-6A13 isalso referred to as insertion site 149743000, and TTS-6A14 is alsoreferred to as insertion site 148293657.

Table 6A shows that the Trait6A insertion resides at IBM position113.62, PHB position 23.70. Transgenic target sites TTS-6A3 wasidentified from MI1 and an additional 10 TTS sites (TTS-6A5, TTS-6A6,TTS-6A7, TTS-6A8, TTS-6A9, TTS-6A10, TTS-6A11, TTS-6A12, TTS-6A13, andTTS-6A14) near the Trait6A genomic locus of interest were identifiedfrom our MI2 collection.

TABLE 6 A Complex Trait Locus CTL-6A on Chromosome 6 of corn. IBM PHBLOCUS location location UMC1625 86.20 16.72 TTS-6A5 76.75 18.40 TTS-6A677.15 18.50 UMC2196 86.40 18.73 TTS-6A7 91.03 19.90 UMC2312 75.80 21.38BNLG1867 78.30 21.49 PZA03047 84.50 21.68 UMC1229 80.70 22.30 UCK1 96.0022.76 RZ390D(CYB5) 85.50 22.79 MMP20 105.90 22.80 MMP10 110.40 22.83MMP160 101.90 22.86 PHP20528 85.50 23.04 UMC2314 99.30 23.10UAZ232B(SCI) 100.30 23.38 UMC2313 91.90 23.56 TTS-6A8 112.25 23.60TTS-6A9 113.62 23.70 TRAIT 6A 23.70 CDO545 86.90 24.72 PHP20854 87.7024.72 UMC1133 98.60 24.72 UFG69 104.80 24.72 TTS-6A10 114.92 23.80TTS-6A11 114.92 23.80 TTS-6A12 119.31 24.90 TTS-6A13 119.53 25.00 MMP76103.80 25.03 TTS-6A14 120.43 25.50 TTS-6A3 122.77 26.30 Y1 120.50 26.81BNLG1422 121.10 26.87 MMP108B 118.30 26.93 MMP4 116.20 27.18 UMC1006125.00 27.94 RZ444E 123.70 31.76

FIG. 9 shows the location of the insertion sites TTSs of the CTL6Acomplex trait locus in relation to public BACS on the maize physical mapof maize.

TABLE 6 B Confirmation of successful site specific integration atdifferent loci comprising the CTL6A. Yes indicates that the target sitelocus was shown to be capable of site specific integration. IBM PHB SSILocus Location Location Confirmed TTS-6A5 76.75 18.4 Yes TTS-6A6 77.1518.5 Yes TTS-6A7 91.03 19.9 Yes TTS-6A8 112.25 23.6 Yes TTS-6A9 113.6223.7 Yes TTS-6A10 114.92 23.8 Yes TTS-6A11 114.92 23.8 Yes TTS-6A12119.31 24.9 Yes TTS-6A13 119.53 25 Yes TTS-6A14 120.43 25.5 Yes

Site Specific Integration in the Transgenic Target Event TTS-6A3

The transgenic event representing TTS-6A3 (FIG. 5B, table 6 A) wasfurther evaluated by testing its capability for site specificintegration. The FRT sites were first sequence verified as previouslydescribed and confirmed to be intact. Particle bombardment was used asthe method of DNA delivery and the same donor plasmid, PHP27064,containing FRT1 and FRT87 was delivered into heterozygous immatureembryos containing TTS-6A3 for SSI. The methods used to perform SSI werethe same as described in Example 2. In addition, a similar series ofqPCR assays to those listed for Example 2 was used to indicate that SSIhad occurred. These included checking the copy number of each genebetween the FRT sites that originally resided in the target (MO-PAT) anddonor (HT, YFP) plasmids as well as detection assays for the otherco-bombarded plasmids (flp, zm-odp2, zm-wus). Assays specific to thenewly formed, recombinant target locus which span the FRT sites are keyto the determination that SSI had occurred. Methods used for the realtime quantitative PCR analysis are the same as those listed for Example2.

There were approximately 4500 immature embryos heterozygous for TTS-6A3,that were co-bombarded using the donor plasmid, PHP27064, and the SSIassociated plasmids with FLP recombinase, ODP2, and WUS (FIG. 7).Bombarded embryos were transferred to selective culture medium withglyphosate as described above and then cultured for callus growth.Callus growing in the presence of glyphosate and showing a yellowfluorescent phenotype when viewed under a dissecting microscope with theappropriate light source and filters was advanced for plantregeneration. We were co-processing material from both TTS-6A3 andTTS-6A4 (Table 7) at the time and observed some differences in YFPphenotype where YFP was uniform and relatively strong from all of thecallus events from TTS-6A3, but weaker and mottled in the TTS-6A4transgenic callus (Table 7). A significant number of independenttransgenic calli with the desired phenotype were advanced intoregeneration and regenerated plants were recovered. Leaf tissue fromplants was analyzed by qPCR analysis to show that SSI was achieved andoften in the absence of integration of the assisting plasmids with flp,zm-wus, and zm-odp2 (Table 8). In the final analysis, there were a fewadditional SSI transgenics that dropped out even though the callusshowed the desired phenotype. Some like event number 6 showed SSI butadditional integration of PHP27064 and PHP31729. Looking at just theclean SSI events, including event numbers 1-2, 4, 7, and 9, we were ableto achieve a 0.11% SSI transformation frequency based on starting embryonumbers in the TTS-6A3 locus. These 5 independent RMCE events wererecovered to the greenhouse and set seed and additional molecularanalysis was planned.

TABLE 7 SSI transformation by particle bombardment. Explant RMCE TargetDonor (no.) (no.) Frequency YFP TTS-6A3 PHP27064 4498 9 0.21% High,uniform TTS-6A4 PHP27064 3118 19 0.60% Low, spotted

TABLE 8 qPCR analysis of regenerated plants from glyphosate resistant,yellow fluorescent calli from immature embryo containing TTS-6A3 targetsite. Transformation methods for site specific integration were used tointroduce PHP27064. qPCR analysis was used to detect SSI and thepresence of plasmids including PHP21139, PHP5096, PHP31729, andPHP27064. Callus Plant FRT1, Donor Target Extras (odp2, Event (No.)FRT87 (HT, yfp) (mopat) wus, flp) 1 10 10/10 10/10 single 10/10 NEG10/10 NEG copy 2 5 5/5 5/5 single copy 5/5 NEG 5/5 NEG 3 9 9/9 9/9single copy 9/9 POS 9/9 POS 4 1 1/1 1/1 single copy 1/1 NEG 1/1 NEG 5 22/2 2/2 multi-copy 2/2 POS 2/2 POS (wus) 6 8 8/8 8/8 multi-copy 8/8 NEG8/8 POS (odp2) 7 3 3/3 3/3 single copy 3/3 NEG 3/3 NEG 8 2 0/2 2/2multi-copy 2/2 NEG 2/2 NEG 9 8 8/8 8/8 single copy 8/8 NEG 8/8 NEG POS =qPCR detection positive. NEG = qPCR detection negative. YFP = yellowfluorescent protein (ZS-Yellow1 NI, Clontech) DNA sequence. HT =herbicide tolerance gene. odp2 = maize ovule development protein 2sequence. wus = maize wuschel sequence. FLP = yeast flp recombinasesequence.

In summary, TTS-6A3 was generated using an SSI platform II construct(PHP36678) different from that (PHP36680) used in Example 2 only byinclusion of different trait genes. This transgenic target site wasselected because it was characterized as a single copy T-DNA insertionusing qPCR analysis during the transformation process. Leaf samples fromthe first generation IE-7 transgenic plant were submitted for flankingsequence analysis and the resulting flanking sequence data was used toprovide an assessment of the genomic position of TTS-6A3 very near toTrait6A on chromosome VI. Later mapping data confirmed the chromosome 6location initially estimated using flanking sequence data. IE7 was nextcharacterized for SSI transformation frequency using PHP27064 anddemonstrated a 0.11% RMCE frequency based on initial qPCR analysis.

Generating a Transgenic Plant Containing the Complex Trait Loci 6A(CTL6A)

Building complex trait locus 6A involves crossing or re-transformingtransgenic plants comprising transgenic target sites with eventscomprising various trait genes in the appropriate region on chromosomeVI (as illustrated in FIG. 1). We identified a number of transgenictarget site candidates from maize inbred 1 and maize inbred 2 which werepredicted to be located in the same region as Trait6A (FIG. 5B). Ifthese insertions were not made with desired trait genes for a particularregion, then the desired genes can be inserted via SSI as wasdemonstrated on chromosome 1 for TTS-6A2 and on chromosome 6 forTTS-6A3. Once the desired genes were inserted, then recombination musttake place to physically link the transgenic insertion sites. We beganby working to obtain linkage between TTS-6A3 and Trait6A using meioticrecombination during flowering.

TTS-6A3 was predicted to be 2.6 cM away from Trait6A based on aninternally derived single meiosis map (PHB) and therefore a predictioncan be made that there would be a nearly 100% chance of finding 5recombinants in 500 progeny. The breeding scheme for physical linkageshould work for any 2 insertion sites or genomic points of interest(transgenic insertions, native traits, QTL's, haplotypes, chromosomalregions of interest, etc.), or combinations of the two (Table 3). Thework involving Trait6A and TTS-6A3 was essentially the same as thatdescribed in Example 2 above.

Seed comprising the Trait6A locus and TTS-6A3 were planted in subsequentweekly plantings and the plants matured to flowering stage. In this workwe used qPCR copy number analysis to identify the homozygous individualsfrom segregating populations. At flowering, homozygous plants forTTS-6A3 were used as a pollen source for plants with the Trait6A locus.Ears were harvested from these crosses, dried down, and F1 kernelscollected. The F1 kernels were planted and the resulting plants werescreened as seedlings by qPCR to confirm that both transgenic loci werepresent. Crossing two homozygous transgenic lines insured that each ofthe F1 progeny had both transgenic insertions. F1 TTS-6A3/Trait6Akernels were planted along with kernels of another suitablenon-transgenic line to act as a female in cross pollination. When theplants had developed to flowering stage, transgenic pollen was carriedto the non-transgenic recipient line. F2 kernels from these crosses wereplanted in larger numbers in flats to screen for recombinants betweenTTS-6A3 and Trait6A. A double herbicide screen was used to select plantsthat were both glyphosate and bialaphos resistant. Bialaphos was appliedfirst when the seedlings were only approximately 10 d after germination.At approximately 3 wk after germination, glyphosate was applied. Doubleresistant plants would be potted for recovery and self-pollination todemonstrate the linkage of the two transgenic traits. Two independenttests were completed involving the germination of 500 kernels for doubleherbicide treatment. In the first set of 500, we identified 5 doubleresistant individuals. The second set of 500 plants only yielded 2double resistant individuals. All of the plants resistant to bothbialaphos and glyphosate were grown to flowering and self-pollinated.The progeny of these crosses would segregate in a ratio of onehomozygous to 2 heterozygous to 1 null for the two herbicide resistancetransgenes. A small set was analyzed for this segregation and theresults are included in Table 9. In conclusion, the analysisdemonstrated that the two transgenes were segregating as a singlegenetic locus. This confirmed our ability to link the two loci, TTS-6A3and Trait6A, into a single trait locus on chromosome 6 to create CTL6A.

TABLE 9 Zygosity qPCR analysis of progeny from self-pollinations ofindividual F2 plants resistant to both Round Up ® and Liberty ®herbicides. F2 Plant Seed ID Homozygous Heterozygous Null Chi² 140047413 6 17 9 0.68 2 40047414 4 20 8 3.00 3 40047431 5 18 7 1.46

Additional Development of Complex Trait Loci 6A (CTL6A)

All the transgenic target sites listed in Table 6A for CTL6A wereadvanced into SSI transformation work following initial determination ofinsertion site flanking sequence and FRT site sequence. Repeatsubmission of samples to verify flanking sequence usually resulted inverification of the initial results (data not shown). Sometimes thesecond pass flanking sequence analysis resulted in better qualitysequence data than the original. Seed increases were completed for eachof these lines to facilitate additional testing in the third generation.Third generation seedlings were grown out for additional sampling andanalysis including qPCR, Southern analysis, and ELISA analysis forexpression (FIG. 10). Samples were collected and analyzed usingquantitative ELISA analysis in order to characterize expressionproperties at each insertion site. Larger numbers of samples could betested since these were young plants (approx. V3/V4) growing in flats.Data in FIG. 10 shows that the ELISA levels for GENE 1, GENE2 and PATwere similar at multiple independent insertion sites except for thefirst event which was shown to have a rearranged T-DNA right border (RB)region. Follow up flanking sequence analysis indicated the problem withthe insertion of the first transgenic population. Mature seedlings weresacrificed after sampling to large leaf samples for high quality genomicDNA extraction and Southern analysis. Site Specific integration occurredat all Target Sites listed in Table 6B (TTS6A5-TTS6A14) as describedbelow.

Site Specific Integration of Target Sites TTS6A5-11S6A14 (Table 6B).

Many loci that are part of CTL6A were further characterized by testingtheir capability for site specific integration (SSI) including TTS-6A5,TTS-6A6, TTS-6A7, TTS-6A8, TTS-6A9, TTS-6A10, TTS-6A11, TTS-6A12,TTS-6A13, and TTS-6A14. Using methods for site specific integrationdescribed in Example 1, a construct named PHP46438 was introducedindependently into each of these loci. PHP46438 is similar to PHP27064(FIG. 7) except that it contained a promoterless NPTII as the first genein the construct and a constitutive AM-CYAN1 gene expressed by the maizeubiquitin promoter as the second. Multiple independent candidates wererecovered for PHP46438 insertion at each of the 10 loci, T0 transgenicplants were regenerated from each candidate, and real time qPCR analysiswas used to identify the events where RMCE had occurred. Plants with thebest qPCR profiles were then recovered to the greenhouse, grown tomaturity, and allowed to set seed. Seed of the next (T1) generation weregrown in flats and analyzed by real time qPCR to characterize transgenesegregation to confirm Mendelian inheritance and to identify homozygousfor seed increase. Transgene positive individuals were sampled forfurther molecular analysis including Southern analysis, sequencing, andELISA characterization. These analyses provided the further confirmationof successful RMCE and site specific integration at all the loci ofCTL6A.

Example 4 Production of a Soybean Library Comprising Transgenic TargetSites for Site Specific Integration (SSI)

Methods of generating transgenic plants, seeds or plant cells comprisingvarious transgenic target sites (TTS) with precise transgeneintegrations for site specific integration (SSI) were developed forsoybean. Transgenic target events were produced by biolistic bombardmentmethods and the transgene integrations were evaluated by sequencingflanking genomic DNA borders.

Development of SSI Target Vectors

The target DNA construct QC599 was modified following standard molecularcloning procedures from a previous SSI target DNA QC288 (Li et al.,Plant Physiol. 151:1087-1095, 2009). QC599 carried the same pair ofdissimilar recombination sites FRT1 and FRT87 to enable subsequent sitespecific integration (SSI) transformation using the yeast FLP/FRTsystem. The soybean S-adenosyl methionine synthetase promoter GM-SAMSPRO (U.S. Pat. No. 7217858B2) was used to drive the expression of thehygromycin phosphotransferase (HPT) gene for soybean transformationselection. Open reading frame stop codon sequences ORFSTOP-B (SEQ IDNO:12) and ORFSTOP-A (SEQ IS NO:13) were added to the expressioncassette ends of QC599 to prevent any potential open reading frames frombeing created when QC599A target gene integrated in soybean genome (FIG.11). The target DNA fragment QC599A used for biolistic bombardmenttransformation was released from construct QC599 by AscI restrictionenzyme digestion and purified from agarose gel with a DNA gel extractionkit (QIAGEN®, Valencia, Calif.).

The FRT1 site was placed between the SAMS promoter and HPT coding regionto set up a gene-trapping configuration. The FRT87 site was placeddownstream of the NOS terminator and in front of the ORFSTOP-A at thevery 3′ end of the target gene cassette. During SSI transformationdescribed schematically in FIG. 12, a SSI donor DNA containing apromoterless marker gene plus any trait genes such as a cyan fluorescentprotein gene CFP flanked together by the same dissimilar FRT1 and FRT87sites was introduced by biolistic bombardment into cells containing thetransgenic target gene. DNA recombination between the corresponding FRT1and FRT87 recombination sites of the donor and target DNA will result inthe cassette exchange between the donor and target DNA in the presenceof FLP recombinase, i.e., every components flanked by the FRT1 and FRT87sites are exchanged between the donor and target DNA in a successfulRMCE (Recombinase Mediated Cassette Exchange). As a result, the initialtarget selectable marker gene HPT was replaced by the newly introducedselectable marker gene HRA (mutated acetolactate synthase) from thedonor to enable the RMCE event to tolerate chlorsulfuron. The ORFSTOP-B,SAMS promoter on the 5′ end and the ORFSTOP-A on the 3′ end of thetarget gene are fixed at the target site and cannot be replaced by RMCE.

Development of SSI Transgenic Target Events by Biolistic Transformation

Purified QC599A DNA fragment was transformed to a Pioneer elite soybeancultivar by the method of biolistic bombardment (Klein et al., Nature327:70-73 (1987); U.S. Pat. No. 4,945,050) as described in detail belowto create transgenic target events from which desired SSI target siteswould be identified by molecular assays and sequence analysis.

Soybean cotyledons (˜3 mm in length) were dissected from surfacesterilized, immature seeds and were cultured for 6-10 weeks in the lightat 26° C. on a Murashige and Skoog (MS) media containing 0.7% agar andsupplemented with 10 mg/ml 2,4-D (2,4-Dichlorophenoxyacetic acid).Globular stage somatic embryos, which produced secondary embryos, werethen excised and placed into flasks containing liquid MS mediumsupplemented with 2,4-D (10 mg/ml) and cultured in the light on a rotaryshaker. After repeated selection for clusters of somatic embryos thatmultiplied as early, globular staged embryos, the soybean embryogenicsuspension cultures were maintained in 35 ml liquid media on a rotaryshaker, 150 rpm, at 26° C. with fluorescent lights on a 16:8 hourday/night schedule. Cultures were subcultured every two weeks byinoculating approximately 35 mg of tissue into 35 ml of the same freshliquid MS medium.

Soybean embryogenic suspension cultures were then transformed by themethod of particle gun bombardment using a DuPont Biolistic™ PDS1000/HEinstrument (Bio-Rad Laboratories, Hercules, Calif.). To 50 μl of a 60mg/ml 1.0 mm gold particle suspension were added (in order): 30 μl of 30ng/μl QC599A DNA fragment, 20 μl of 0.1 M spermidine, and 25 μl of 5 MCaCl₂. The particle preparation was then agitated for 3 minutes, spun ina centrifuge for 10 seconds and the supernatant removed. The DNA-coatedparticles were then washed once in 400 μl 100% ethanol and resuspendedin 45 μl of 100% ethanol. The DNA/particle suspension was sonicatedthree times for one second each. Then 5 μl of the DNA-coated goldparticles was loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture was placedin an empty 60×15 mm Petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5 to 10 plates of tissue were bombarded. Membrane rupture pressure wasset at 1100 psi and the chamber was evacuated to a vacuum of 28 inchesmercury. The tissue was placed approximately 3.5 inches away from theretaining screen and bombarded once. Following bombardment, the tissuewas divided in half and placed back into liquid media and cultured asdescribed above.

Five to seven days post bombardment, the liquid media was exchanged withfresh media containing 30 μg/ml hygromycin B as selection agent. Thisselective media was refreshed weekly. Seven to eight weeks postbombardment, green, transformed tissue was observed growing fromuntransformed, necrotic embryogenic clusters. Isolated green tissue wasremoved and inoculated into individual flasks to generate new, clonallypropagated, transformed embryogenic suspension cultures. Each clonallypropagated culture was treated as an independent transformation eventand subcultured in the same liquid MS media supplemented with 2,4-D (10mg/ml) and 30 μg/ml hygromycin B selection agent to increase mass. Theembryogenic suspension cultures were then transferred to agar solid MSmedia plates without 2,4-D supplement to allow somatic embryos todevelop. A sample of each event was collected at this stage forquantitative PCR analysis.

Cotyledon stage somatic embryos were dried-down (by transferring theminto an empty small Petri dish that was seated on top of a 10 cm Petridish containing some agar gel to allow slow dry down) to mimic the laststages of soybean seed development. Dried-down embryos were placed ongermination solid media and transgenic soybean plantlets wereregenerated. The transgenic plants were then transferred to soil andmaintained in growth chambers for seed production.

Identification of SSI Transgenic Target Sites

Transgenic events sampled at early somatic stage were analyzed by realtime quantitative PCR (qPCR) to assess the copy numbers of the QC599Atransgenic target gene and then by inverse PCR to sequence the genomicDNA borders flanking the QC599A transgene of only the single copy eventsidentified by the qPCR.

Genomic DNA were extracted from somatic embryo samples and analyzed byquantitative PCR using the 7500 real time PCR system (AppliedBiosystems) with gene-specific primers and fluorescence probes to checkcopy numbers around both the FRT1 and FRT87 sites of transgenic QC599A.The qPCR analysis was done in duplex reactions with a heat shock protein(HSP) gene as the endogenous controls and a transgenic DNA sample with aknown single copy of QC599A transgene as the calibrator using therelative quantification methodology (Applied Biosystems). The endogenouscontrol HSP probe was labeled with VIC and the target gene probes werelabeled with FAM for the simultaneous detection of both fluorescentprobes. Only events identified to be single copy by both the FRT1 andFRT87 qPCR assays were further analyzed and advanced to regenerateplants.

The same genomic DNA used for qPCR of selected single copy events werefragmented with three different restriction enzymes AflII, NsiI, andPciI that all cut QC599A only once and all in the NOS region so commonprimers could be used for inverse PCR (FIG. 13). For example, PciI cuttransgene QC599A once in the NOS region and once in the flanking genomicDNA border nearest to the 5′ end of QC599A to create a DNA fragmentcontaining a genomic DNA segment bordering the 5′ part of QC599A and the5′ portion of transgenic QC599A with PciI half site on both the 5′ and3′ ends. Similarly, a DNA fragment containing the 3′ portion oftransgenic QC599A and a genomic DNA segment bordering the 3′ end ofQC599A was simultaneously created also ending with PciI half site onboth 5′ and 3′ ends. A self ligation step then circulized both the 5′border and 3′ border fragments making their genomic DNA portionsamplifiable by PCR using only QC599A-specific primers

Two rounds of PCR amplification were applied to minimize theamplification of any non-specific genomic DNA fragments (FIG. 13).Invitrogen high fidelity Taq DNA polymerase was used to amplify thedigested and then self-ligated genomic DNA templates 25 cycles by thefirst PCR. Then 1% of each first PCR product was amplified 35 cycles bythe second PCR. The second PCR products were checked by agarose gelelectrophoresis and most of the times distinct bands were amplified. ThePCR product displayed a distinct band was then sequenced using theSanger method with the same corresponding sense and antisense primersused in the second PCR. Sequences were assembled and analyzed usingprograms in the Vector NTI suites (Invitrogen). Sequences were used toBLAST search the Joint Genome Institute soybean genome sequencespublically available to identify their locations in the genome bysequence homology.

Total of 830 QC599A transgenic events were produced and analyzed forcopy numbers by qPCR and 360, up to 43% of the total, were selected assingle copy events for border sequencing by inverse PCR (Table 10). Both5′ and 3′ border sequences were obtained from 239 of the single copyevents. But the majority of them have a 5′ border and a 3′ border thatare often from different chromosomes or are not naturally continuouseven when they are on the same chromosome. Only 19 of the sequenced 239events have their 5′ border and 3′ border on the same chromosome andalso are naturally continuous. Some of them may have small deletions orinsertions at the transgenic QC599A insertion site. The 19 events areconsidered as quality events with matching genomic DNA borders andselected as SSI target lines to be used for future SSI transformation.

TABLE 10 Soybean SSI target events evaluation. Sample Description SampleNo. Total events submitted for copy number check by qPCR 830 Single copyevents selected for inverse PCR 360 Events with 5′ border inverse PCRbands 313 Events with 3′ border inverse PCR bands gene 310 Events withboth 5′ and 3′ border inverse PCR bands 274 Events with 5′ bordersequences 281 Events with 3′ border sequences 247 Events with both 5′and 3′ border sequences 239 Events with matching 5′ and 3′ bordersequences 19

When an SSI transgenic target site was identified in a genomic locus ofinterest in the soybean genome then the regenerated T0 plant wasanalyzed by qPCR and Southern hybridization to confirm previous copynumber check and border sequencing results. Multiple digestions such asNdeI, NsiI, and PciI and different probes specific to the SAMS promoter,HPT coding region, and flanking genomic borders were used in theSouthern analysis and all the bands had to match the sizes predictedfrom the genomic DNA sequences surrounding the insertion site in orderto confirm the target site.

Transgenic SSI target T0 plants were maintained in controlled growthchambers and monitored for agronomic abnormality until T1 seeds wereharvested. Sixty four T1 seeds of each target event were planted and T1plants were analyzed by the same FRT1 and FRT87 qPCR assays to checktransgenic QC599A segregation. Selected homozygous T1 plants were thensampled and analyzed by similar Southern hybridization to confirmprevious Southern results obtained on the T0 plant. Only after passingall the above evaluations, a SSI transgenic target event containing atransgenic target site was advanced as a recombination target locus(RTL) that could be used as a target line in SSI transformation.

Example 5 Production of a Complex Trait Locus on Chromosome 19

One valuable region of the soybean genome, referred to as Complex TraitLocus LA (CTL-LA), was identified containing a trait of interest(Trait-LA) on chromosome 19 (or linkage group L) and at least two SSItarget sites in its proximity (FIG. 14).

Identification of Complex Trait Locus

The location of the trait of interest Trait-LA on chromosome 19 wasdetermined based on sequence homology following the determination oftransgenic target sites flanking sequences and then verified by physicaland genetic mapping data. Transgenic target site flanking sequences (FS)were obtained by using inverse PCR. Each FS sequence was compared to thesoybean whole genome sequence using the BLAST algorithm to predict itslocation to a chromosome since most soybean genome sequences have beenassembled into 20 individual chromosomes (DNA markers close to the FSlocation identified by sequence continuity were then used to associatethe FS location, which represents the transgenic SSI target sitelocation, to the soybean genetic map. Finally, the genetic map positionof the transgenic SSI target site was used to determine if it is closeto a trait gene of interest. As an example in this application, twotransgenic SSI target sites TTS-LA1 and TTS-LA2 were found to be in theproximity of the trait gene of interest Trait-LA. In conclusion, acomplex trait locus named CTL-LA was created containing the native traitgene LA locus and two nearby transgenic SSI target loci TTS-LA1 adTTS-LA2 (FIG. 14). The independent transgenic loci can then be broughttogether to the native trait locus from different parents by breedingand then the complex trait locus can segregate in subsequent breedingsteps as a single genetic locus.

TABLE 11 Complex trait locus CTL-LA on chromosome 19 of soybean. SoybeanComposite Pioneer Soy LOCUS Public map v1.2 SATT613 36.04 45.1 SATT28438.16 47.7 S60414-TB 49.3 SATT462 41.0 49.3 TTS-LA1 57.2 TRAIT-LA 60.1TTS-LA2 62.9 SATT481 54.57 65.8 SATT156 56.13 65.8 SCT_010 59.52 68.5

In the process of developing a soybean TTS library, two transgenic SSItarget sites TTS-LA1 and TTS-LA2 were identified on Chromosome 19 near atrait of interest Trait-LA. The insertion site of TTS-LA1 was determinedto be only approximately 2.9 cM (centi-Morgan) close to a trait ofinterest (Trait-LA) mapped at position 60.1 cM on chromosome 19 ofproprietary Pioneer soybean map v1.2 (Table 11) while TTS-LA2 wasapproximately 2.8 cM close to the Trait-LA on the opposite side ofTTS-LA1. More genetic markers and associated trait genes withinapproximately 10 cM of the Trait-LA locus are obvious from the map andsome of them are listed in Table 11. The corresponding positions of someof the same markers on the soybean composite public map were determinedby searching the markers on the public map (Table 11).

Site Specific Integration at Transgenic Target Sites TTS-LA1 and TTS-LA2

The transgenic events containing either the TTS-LA1 or the TTS-LA2 siteswere further evaluated by testing its capability for site specificintegration of genes of interest. T0 transgenic plants of the two eventswere brought to maturity and T1 seeds were planted in controlled growthchambers. Leaf samples of T1 transgenic plants were first analyzed byFRT1 and FRT87 qPCR to confirm that the single copy QC599A DNA fragmentsegregate Mendelian inheritance and to identify homozygous plants.Selected homozygous T1 plants were analyzed by Southern hybridization toconfirm that the hybridized bands match the sizes predicted from thegenomic DNA sequence of the chromosome 19 segment harboring the QC599Atransgene. A part of the 5′ border, the entire QC599A DNA fragment, anda part of the 3′ border were also amplified by PCR using a 5′ bordersense primer and a 3′ border antisense primer and sequenced to confirmboth the transgene and the border junctions to be intact.

Developing T2 embryos were excised from the homozygous T1 plants andused as explants to initiate tissue cultures which were then transformedwith a SSI donor DNA construct with the help of a FLP expressionconstruct by biolistic bombardment as described in EXAMPLE 4. One targetsite TTS-LA1 was transformed with a SSI donor DNA construct QC728containing one or more trait genes of interest. FLP recombinase activitywas provided by the transient expression of the cobombarded DNAconstruct QC663. Only SSI transgenic events in which the GM-ALS markergene of the donor QC728 was placed through FLP recombinase mediated DNArecombination downstream of the GM-SAMS promoter of the QC599A transgeneat the TTS-LA1 target site were selected with 90 ng/ml chlorsulfuron.Transgenic events were sampled at callus stage and analyzed by a seriesof qPCR assays to determine if SSI had occurred and RMCE events wereproduced in a manner similar as depicted in FIG. 12. Different traitssuch as herbicide tolerance genes were integrated at the other SSItarget site TTS-LA2 following similar transformation procedure usingdifferent donor DNA constructs and the transgenic events were analyzedby similar qPCR assays.

Since the SSI target events containing QC599A DNA fragment weretransformed with a donor DNA in the presence of a FLP expression DNAconstruct to produce a RMCE through FLP mediated recombination, four DNAincluding the target QC599A, the donor QC728, the FLP expressionconstruct QC663, and the RMCE product QC599A728A could coexist in anytransgenic events that passed chlorsulfuron selection. Randomlyintegrated DNA can be segregated away from the RMCE locus to generate aclean RMCE event comprising a trait of interest at one locus (integratedat either TTS-LA1 or TTSLA2). Cumulative qPCR results showed that SSIhad occurred and RMCE events were recovered from both TTS-LA1 andTTS-LA2 transformation.

Transgenic events comprising a RMCE at TTS-LA1 can be crossed withtransgenic events comprising a RMCE at TTS-LA2 to generate a complextrait loci comprising stacked traits of interest.

Generating a Transgenic Plant Containing the Complex Trait Locus CTL-LA

In order to build the complex trait locus CTL-LA, transgenic plantscarrying trait genes at TTS-LA1 site need to be crossed with transgenicplants carrying different trait genes at TTS-LA2 site (FIG. 14). SinceTTS-LA1 site is 2.9 cM away from Trait-LA and TTS-LA2 site is 2.8 cM ofTrait-LA, the total distance between TTS-LA1 and TTS-LA2 sites is 5.8cM. When two RMCE events with trait genes at each the TTS-LA1 andTTS-LA2 sites are crossed, there is close to 6% chance of finding aprogeny carrying the traits at both sites after only one generationsince one cM is equal to a 1% chance that a marker at one genetic locuswill be separated from a marker at another locus due to crossing over ina single generation.

Selected RMCE events containing the trait gene of interest at TTS-LA1can be brought to maturity and their homozygous T1 plants can beidentified by qPCR, evaluated by Southern hybridization and phenotypicanalysis. One perfect RMCE event can be selected as a parent. Anotherparent can be selected from RMCE events with, for example, herbicidetolerance traits at TTS-LA2 site through similar qPCR, Southernhybridization, and phenotypic analysis. The two parents can be crossedand the resulting progenies can be analyzed by qPCR assays specific toeach parent to identify recombinants that contain both TTS-LA1, TTS-LA2,traits as well as the native trait Trait-LA between them (FIG. 14). F1seeds of the identified recombinants can be planted and T2 progenieshomozygous for the TTS-LA1-Trait-LA-TTS-LA2 complex trait locus CTL-LAcan be produced when the plants are naturally self-pollinated. Thehomozygous TTS-LA1-Trait-LA-TTS-LA2 complex trait locus can beidentified by similar qPCR assays and can be propagated subsequently asa single locus.

TABLE 12 Summary of SEQ ID NOS. SEQ ID NO: NT/AA Description 1 NTMinimal FRT1 recombination site 2 NT Minimal FRT5 recombination site 3NT Minimal FRT6 recombination site 4 NT Minimal FRT7 recombination site5 NT Minimal FRT12 recombination site 6 NT Minimal FRT87 recombinationsite 7 NT Cre recombinase 8 NT FLP recombinase 9 NT Variant of Crerecombinase (moCre-maize preferred codons) 10 NT Variant of FLPrecombinase (FLPm-maize preferred codons) 11 NT SSI target DNA fragmentQC599A used for biolistic soybean transformation to create transgenictarget events. 12 NT An oligonucleotide containing stop codons in allsix possible open reading frames- ORFSTOP-B. 13 NT Anotheroligonucleotide containing stop codons in all six possible open readingframes- ORFSTOP-A.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. A maize plant or seed having in its genome agenomic window comprising at least a first transgenic target sitecomprising at least two non-identical FRT recombination sites, a secondtransgenic target site comprising at least two non-identical FRTrecombination sites, and a genomic locus of interest; wherein each ofsaid genomic locus of interest, said first transgenic target site andsaid second transgenic target site have different genomic sites; andwherein the genomic window is flanked by a first marker and a secondmarker; wherein the first marker is UMC2196, and the second marker isUMC1006.
 2. The plant or seed of claim 1, wherein the non-identical FRTrecombination sites of the first transgenic target site comprise a FRT1site, a FRT5 site, a FRT6 site, a FRT7 site, a FRT12 site, or a FRT87site.
 3. The plant or seed of claim 1, wherein the non-identical FRTrecombination sites of the second transgenic target site comprise a FRT1site, a FRT5 site, a FRT6 site, a FRT7 site, a FRT12 site, or a FRT87site.
 4. The plant or seed of claim 1, wherein the non-identical FRTrecombination sites of the first transgenic target site comprise a FRT1site and a FRT87 site.
 5. The plant or seed of claim 1, wherein thenon-identical FRT recombination sites of second first transgenic targetsite comprise a FRT1 site and a FRT87 site.
 6. The plant or seed ofclaim 1, wherein the non-identical FRT recombination sites of the firsttransgenic target site comprise a FRT1 site and a FRT6 site.
 7. Theplant or seed of claim 1, wherein the non-identical FRT recombinationsites of second transgenic target site comprise a FRT1 site and a FRT6site.
 8. The plant or seed of claim 1, wherein said genomic windowfurther comprises a third transgenic target site comprising a fourth FRTrecombination site, a fifth FRT recombination site and a sixth FRTrecombination site, wherein (i) said fourth, fifth and said sixth FRTrecombination sites are dissimilar with respect to one another; or (ii)said fourth, fifth and said sixth FRT recombination sites are dissimilarand have a decreased compatibility with respect to one another; and saidthird transgenic target site has a different genomic insertion site thansaid first transgenic target site, said second transgenic target siteand said genomic locus of interest.
 9. The maize plant or seed of claim1, wherein (a) said genomic locus of interest confers a trait comprisingmale sterility, site-specific recombination, abiotic stress tolerance,altered phosphorus, altered antioxidants, altered fatty acids, alteredessential amino acids, altered carbohydrates, herbicide tolerance,insect resistance or disease resistance; or (b) said genomic locus ofinterest comprises a transgene or a native trait.
 10. The plant or seedof claim 1, wherein said first transgenic target site or said secondtransgenic target site comprises at least one polynucleotide ofinterest.
 11. A method for producing a second plant, the methodcomprising applying plant breeding techniques to a first plant, or partsthereof, wherein said first plant is the plant of claim 1, and whereinapplication of said techniques results in the production of said secondplant, wherein said second plant comprises at least one additionaltransgenic target site or at least one additional genomic locus ofinterest within said genomic window when compared to said first plant;wherein each of said additional transgenic target site and saidadditional genomic locus of interest have a different genomic insertionsite with respect to each other and with respect to said firsttransgenic target site, said second transgenic target site and saidgenomic locus of interest.
 12. The method of claim 11, wherein the atleast one additional transgenic target site comprises a polynucleotideof interest.
 13. The method of claim 11, wherein said second plantcomprises at least one less transgenic target site or at least one lessgenomic locus of interest within said genomic window when compared tosaid first plant.
 14. A method of producing a complex trait locus in thegenome of a plant, the method comprising: (a) providing a first planthaving in its genome a genomic window comprising at least a firsttransgenic target site comprising at least two non-identical FRTrecombination sites, and wherein said first plant does not comprise afirst genomic locus of interest; wherein the genomic window is flankedby a first marker and a second marker; wherein the first marker isUMC2196, and the second marker is UMC1006; (b) breeding to said firstplant a second plant comprising at least two non-identical FRTrecombination sites, wherein said second plant comprises in its genomethe first genomic locus of interest and said second plant does notcomprise said first transgenic target site; and, (c) selecting a progenyplant from step (b) comprising said first transgenic target site andsaid genomic locus of interest; wherein said first transgenic targetsite and said first genomic locus of interest have different genomicinsertion sites in said progeny plant.
 15. A method of producing acomplex trait locus in the genome of a plant, the method comprising: (a)providing a first plant having in its genome a genomic window at least afirst transgenic target site and a second transgenic target site havingdifferent genomic insertion sites, wherein said first transgenic targetsite comprises at least two non-identical FRT recombination sites andsaid second transgenic target site comprises at least two non-identicalFRT recombination sites, wherein said first plant does not comprise afirst genomic locus of interest; wherein the genomic window is flankedby a first marker and a second marker; wherein the first marker isUMC2196, and the second marker is UMC1006; (b) breeding to said firstplant a second plant, wherein said second plant comprises in its genomethe first genomic locus of interest, wherein said second plant does notcomprise said first transgenic target site or said second transgenictarget site; and, (c) selecting a progeny plant from step (b) comprisingsaid first transgenic target site, said second transgenic target siteand said first genomic locus of interest; wherein each of said firsttransgenic target site, said second transgenic target site and saidfirst genomic locus of interest have a different genomic insertion sitein said progeny plant.
 16. The method of claim 15, wherein said methodfurther comprises (a) breeding to said progeny plant a third plantcomprising a second genomic locus of interest, wherein said third plantcomprises in said genomic window said second genomic locus of interest,wherein said third plant does not comprise said first transgenic targetsite, said second transgenic target site or said first genomic locus ofinterest in said genomic window; and, (b) selecting a second progenyplant from step (a) comprising said first transgenic target site, saidsecond transgenic target site, said first genomic locus of interest, andsaid second genomic locus of interest; wherein each of said firsttransgenic target site, said second transgenic target site, said firstgenomic locus of interest and said second genomic locus of interest havea different genomic insertion site in said second progeny plant.
 17. Themethod of claim 15, wherein (a) the first genomic locus of interestconfers a trait comprising male sterility, site-specific recombination,abiotic stress tolerance, altered phosphorus, altered antioxidants,altered fatty acids, altered essential amino acids, alteredcarbohydrates, herbicide tolerance, insect resistance or diseaseresistance; or (b) the first genomic locus of interest comprises anative trait locus, a transgene of interest, or an additional transgenictarget site.
 18. The method of claim 15, wherein (a) said firsttransgenic target site comprises a first FRT recombination site and asecond FRT recombination site, wherein (i) said first and said secondFRT recombination sites are dissimilar with respect to one another and,said first transgenic target site comprises a polynucleotide ofinterest; or (ii) said first and said second FRT recombination sites arenon-identical and have a decreased compatibility with respect to oneanother and, said first transgenic target site comprises apolynucleotide of interest; and, (b) said second transgenic target sitecomprises a third FRT recombination site and a fourth FRT recombinationsite, wherein (i) said third and said fourth FRT recombination sites arenon-identical with respect to one another; and said second transgenictarget site further comprises a second polynucleotide of interest; or(ii) said third and said fourth FRT recombination sites arenon-identical and have a decreased compatibility with respect to oneanother; and said second transgenic target site further comprises asecond polynucleotide of interest.
 19. The method of claim 18, whereinsaid first transgenic target site and said second transgenic target sitecomprise (a) the same non-identical FRT recombination sites; or (b)different non-identical FRT recombination sites.
 20. The method of claim18, wherein the first and second FRT recombination sites comprise a FRT1site, a FRT5 site, a FRT6 site, a FRT7 site, a FRT12 site, or a FRT87site.
 21. The method of claim 18, wherein the third and fourth FRTrecombination sites comprise a FRT1 site, a FRT5 site, a FRT6 site, aFRT7 site, a FRT12 site, or a FRT87 site.
 22. The method of claim 18,wherein the first and second FRT recombination sites comprise a FRT1site and a FRT87 site.
 23. The method of claim 18, wherein the third andfourth FRT recombination sites comprise a FRT1 site and a FRT87 site.24. The method of claim 18, wherein the first and second FRTrecombination sites comprise a FRT1 site and a FRT6 site.
 25. The methodof claim 18, wherein the third and fourth FRT recombination sitescomprise a FRT1 site and a FRT6 site.